International Journal of Cancer (IJC), we provide an overview of the issues pertinent to misidentified cell lines and discuss available solutions. We also summarize the lessons learned, revealing that at least 5% of the human cell lines used in manuscripts considered for peer review are misidentified. About 4% of the considered manuscripts are rejected for severe cell line problems, and most are subsequently published in other journals. In order to diminish such malpractice and its consequences for the scientific record, we postulate that strict multi‐layered quality control is essential. Besides journals and publishers, we encourage scientists, research institutions, and funders to take action on the matter and revise their respective policies. Hence, we provide concrete recommendations on introducing regular authentication schemes and staff training, and discuss future steps for enhancing good cell culture practices.Immortalized or continuous cell lines are invaluable tools in basic and preclinical research. However, the widespread use of misidentified cell lines is a serious threat to scientific reproducibility. Based on the experiences of mandatory cell line authentication at the  This commentary discusses multi‐layered quality control of immortalized cell lines as an essential prerequisite for reliability of the scientific record and good cell culture practices. Philippe Gorphe identified 1,036 articles published between 1954 and January 2018 specifically referring to the wrong laryngeal origin of HEp‐2 cells. He also showed that the number of manuscripts using the HEp‐2 cell line as a laryngeal carcinoma has increased over the past three decades , cell line cross‐contamination decreased from 15 to 6%. However, among cell lines obtained from secondary sources , the cross‐contamination prevalence remained high at 14–18% over the 25‐year timespan, indicating that one in six of the secondarily sourced cell lines, shared between laboratories, is misidentified , including 848 leukemia–lymphoma cell lines received between 1990 and 2014 from 290 laboratories in 23 countries, showed a difference in the prevalence of cell line cross‐contamination depending on the cell line source. Among cell lines obtained from primary sources  raised alarm bells when they reported a misidentified cell line rate of 25% after profiling 380 cell lines obtained from 113 independent sources , the corresponding purchase orders or invoices are accepted for authentication.Of continuous human cell lines obtained within the last 3 years from a commercial source, which guarantees cell line authenticity through in‐house quality control measures , and should show that the cell line is unique and not cross‐contaminated or misidentified.The •IJC also accepts single nucleotide polymorphism (SNP) based cell line authentication reports from service providers with certified quality control, but only for cell lines of which an SNP‐based reference profile is publicly available.The •Authors of studies describing the establishment of new human cell lines are strongly encouraged to include the summarized STR results in the manuscript for future reference.•○e.g., HeLa (RRID:CVCL_0030)).All cell lines used must be listed using the official cell line name and its Research Resource Identifier (RRID) as available in the ExPASy Cellosaurus database  profiling within the last 3 years.○A statement confirming that all experiments were performed with mycoplasma‐free cells.Since May 2019, the following information must be included in the Materials and Methods section:aAdapted from https://onlinelibrary.wiley.com/page/journal/10970215/homepage/ForAuthors.html#AUCEL.bUntil May 2019 cell line authentication documents not older than 4 years were accepted.In this 3‐year period, 747 manuscripts containing original data on 4,138 human cell lines were considered for peer review. The majority of these submissions (∼65%) were not accompanied by valid cell line authentication documents and had to be unsubmitted to ask for correct documents, indicating that regular authentication of cell lines is not common practice. Eventually, valid authentication documents were obtained from 3,091 (74.7%) cell lines. By verifying the available cell line authentication documentation and cross‐checking the Cellosaurus database, 216 (5.2%) misidentified cell lines were identified, including 186 misidentified cell lines that were completely overgrown and replaced by a contaminator cell line, 16 mixtures of two cell lines, and 14 reported nonintentional hybrid cell lines Fig . The moset al, The highest percentage of misidentified cell lines was observed among gastric cancer (25.4%), followed by liver cancer 16%) cell lines Fig . Hence, 6% cell le.g., manipulated electropherograms or fake invoices).Based on the available information, no cell line‐related problems were observed in 77.1% of the considered manuscripts Fig . HoweverBox 2•No cell line problems: Based on the available cell line authentication documentation and by cross‐checking the Cellosaurus database, no cell line issues were observed.•Minor cell line problems: These manuscripts only needed minor textual clarifications. For instance, the authors used a cell line that is known to be misidentified by another cell line from the same tumor type, as recorded in the Cellosaurus database, and the authors were asked to add this information to their manuscript . Or the authors were asked to clarify that two of the used cell lines originate from the same donor .•Moderate cell line problems: One cell line had to be removed from the manuscript because it was misidentified by a cell line from a different tumor type, the authentication documents revealed that the sample contained a mixture of two cell lines, or the cell line is recorded in Cellosaurus as a reported nonintentional hybrid cell line. Moderate cell line issues also include manuscripts containing misidentified cell lines that did not need to be removed because they are known to be misidentified by a cell line from the same tumor type as recorded in Cellosaurus, but the misidentified cell line had a large impact on the significance of the manuscript . Moderate cell line issues also include manuscripts in which a cell line needed to be renamed because the authentication documents showed that the cell line was misidentified by another cell line from the same tumor type, but the cell line was not among the known misidentified cell lines recorded in Cellosaurus.•Severe cell line problems: At least two cell lines had to be removed from the manuscript because they were misidentified by a cell line from a different tumor type, the samples contained a mixture of two cell lines, and/or reported nonintentional hybrid cell lines were used. In case the misidentified cell line(s) affected a large amount of the presented data and/or the cell line authentication documentation was clearly falsified , then it was decided to reject the manuscript for severe cell line issues.The cell line‐related problems observed in the manuscripts that were considered for peer review and included original human cell line data were divided into the following categories:IJC with severe cell line‐related problems are subsequently published elsewhere, we followed up 63 manuscripts. These included 39 manuscripts from the analysis above that had been considered for peer review but were unsubmitted or rejected for serious cell line‐related problems between July 2018 and December 2020. The remaining 24 manuscripts that we followed up from this time period were not considered for peer review, but it was noticed that a large amount of the data were based on (known) misidentified cell lines. In all cases, the authors were clearly informed about the cell line problems observed in their manuscript in the unsubmission or rejection letter. Afterwards, we observed that 50 (79.4%) of these manuscripts were published in other journals, of which 45 (71.4%) manuscripts still included the data of the misidentified cell lines. Of these 45, 32 (50.8%) manuscripts included data of known misidentified cell lines with no known authentic stock, which means that the existing cell line problems could not have been solved in the meanwhile. Remarkably, in 5 (7.9%) cases, the data of known misidentified cell lines with no known authentic stock was renamed into another cell line. Additionally, one such manuscript was even resubmitted to the IJC. As the intentional publishing of misidentified cell line data represents scientific misconduct, the policy of the IJC is that new submissions of authors rendered guilty in this regard will not be considered. Because of data protection rules, the IJC cannot share personal data of authors with other journals. However, in order to correct the records, other journals are invited to contact the IJC to obtain general instructions on how they can identify published manuscripts that are based on known misidentified cell lines.In order to explore whether manuscripts rejected at the Our experiences show that the misidentified cell line problem is very persistent and that strict editorial quality control procedures are essential to prevent publishing data based on misidentified cell lines. In order to avoid the publication of studies including misidentified cell line data and to correct the contaminated published records, journals and publishers should play a more active role. Hence, specific recommendations and perspectives for journals and publishers are discussed below and summarized in Fig et al, Many commonly used cell line names are not unique Bairoch, . Hence, Moreover, about 60% of the misidentified cell lines identified in our evaluation are (now) known misidentified cell lines and are recorded in the Cellosaurus database. Therefore, journals can easily prevent publishing data based on these problematic cell lines by verifying all cell lines used in a manuscript in the Cellosaurus database even before starting the peer review process.Around 40% of the misidentified cell lines identified in our evaluation are not listed as known misidentified cell lines in the Cellosaurus database. This portion of misidentified cell lines can thus only be uncovered by implementing a strict mandatory cell line authentication policy. Verification of the authentication data by a qualified person is crucial because misidentified cell lines are often missed by authors due to misinterpretation of the STR results. We realize that verification of all cell line authentication documents will not be feasible for every journal. Nevertheless, if journals implement mandatory cell line authentication in combination with occasional verification of the documents, regular authentication of cell lines will become common practice and misidentified cell lines will be identified and eliminated earlier.Even a strict cell line policy cannot completely prevent publishing manuscripts based on misidentified cell lines, and we encountered several cases where a correction of already published manuscripts with cell line problems became necessary.et al, Exemplarily, we obtained a unique STR profile of the presumed lung cancer cell line SPC‐A1, of which no reference STR profile was publicly available and the obtained STR profile did not give a match in the available STR databases. Two years later, we reassessed the STR profile in the updated Cellosaurus CLASTR tool, which revealed that the used SPC‐A1 cell line was actually the hepatocellular carcinoma MHCC97‐H cell line , sometimes even accompanied by misleading cell line authentication reports. At the IJC, we accept purchase orders or invoices of authors who purchased their cell lines not longer than 3 years ago from commercial sources guaranteeing cell line authenticity through in‐house quality control measures, assuming that these sources would not distribute misidentified cell lines. Although our procedure has been adapted and invoices of such doubtful sources are now only accepted together with valid certificates of authentication, in the past manuscripts using these misidentified cell lines have been published and we are now in the process of correcting these records. However, publishing errata is a time‐consuming process, and there are hundreds of thousands of manuscripts published in other journals, based on known misidentified cell lines with no known authentic stock, that need to be corrected. Therefore, we support the idea of Horbach & Halffman  as Supplementary Material.Nowadays, many biomedical journals, including the IJC also receives studies that use cell lines of nonhuman origin, among which mouse cell lines are the most common. Although the species of origin and inter‐species cross‐contamination can easily be detected using a multiplex PCR‐based assay , and whenever a phenotypic change—particularly in growth behavior—is observed. In general, scientists are advised to acquire continuous cell lines directly from certified cell banks that guarantee cell line authenticity through internal STR analyses . If a cell line is obtained from a secondary source , the cell line should be quarantined antibiotic‐free until its identity has been confirmed by STR profiling, and the overall sterility and absence of mycoplasma have been verified.Cell lines in long‐term use should be authenticated at the beginning and the end of a project, especially after selection procedures . For future reference, summarized STR results should be included in the manuscript as an essential part of the initial description so that authentication data can be included in the Cellosaurus database and associated with the RRID.The establishment of new cell line models is an important but difficult task, as cross‐contamination at the source is one of the most common causes for the development of misidentified cell lines (MacLeod https://iclac.org/education/). For further and detailed instructions on good cell culture practice, we refer to the “Guidance document on good in vitro method practices (GIVIMP)” by the Organization for Economic Co‐operation and Development , the research institutions themselves, and finally, the publication bodies. In this respect, funding agencies can play a very important role if they require proof of authentication for the planned use of tumor models to be submitted at the time of application, as already practiced by the German Wilhelm Sander Foundation, the US National Institutes of Health, or the British Cancer Research UK.IJC, we notice that authentication of cell lines is a more common practice among researchers who are affiliated to research institutions with an own dedicated cell line authentication facility. This type of authentication infrastructure has long been established at large companies such as AMGEN, Merck, or Roche Holding, but is also increasingly provided at large research institutions such as the German DKFZ or MD Anderson Cancer Center in the USA. Internal STR profiling facilities allow easy access to rapid authentication testing and ensure the presence of in‐house expertise for the interpretation of challenging samples, such as hybrid cell lines or mixtures. Hence, research institutions should consider offering a cell line authentication service in their own core facility. In addition, biomedical research institutions should develop guidelines for cell line use, as implemented by the MD Anderson Cancer Center requiring all researchers to authenticate their cell lines once a year. For a draft, we refer the reader to Schweppe and Korch, who suggested a detailed cell line and tissue authentication policy for biomedical research institutions (Schweppe & Korch, At the “This analysis could become a prerequisite for publication, so that the problem of cell line cross‐contamination can be reduced to a minimum in the future” (Masters et al, IJC, still at least 5% of the human cell lines used in manuscripts considered for peer review are misidentified and about 4% of the considered manuscripts are rejected for severe cell line problems. The fact that the majority of the manuscripts unsubmitted or rejected for severe cell line problems have since been published in other journals, illustrates the irresponsibility of some authors toward the misidentified cell line problem. Hence, we call on scientists, research institutions, journals, and publishers to take action by implementing a mandatory cell line authentication policy and by developing a strategy to correct the contaminated literature. A collaborative approach that includes proactive funding agencies, rapid access to low‐cost authentication services, and widely requested proof of authentication from journals should significantly reduce the chronic dilemma of using misidentified cell lines in the future.Twenty years ago, when John Masters and colleagues introduced STR profiling as the international reference method for human cell line authentication, they were very optimistic and stated: Nicole Y Souren: Conceptualization; data curation; formal analysis; investigation; visualization; writing – original draft; writing – review & editing. Norbert E Fusenig: Conceptualization; supervision; writing – review & editing. Stefanie Heck: Conceptualization; data curation; investigation; writing – review & editing. Wilhelm G Dirks: Expert advice; visualization; writing – review & editing. Amanda Capes‐Davis: Expert advice; writing – review & editing. Franca Bianchini: Supervision; project administration; writing – review & editing. Christoph Plass: Conceptualization; supervision; project administration; writing – review & editing.The authors declare that they have no conflict of interest.


Effective alternative strategies and methodological approaches are critically necessary for cancer prevention and therapy. In this study, we investigated the antitumor potential of neem fruit mesocarp and epicarp extracts. The chemical composition of the derived extracts was characterized using GC–MS. Data were collected on the antimicrobial activity of the extracts in addition to the cytotoxicity effect evaluated against PC-3, MCF-7, and Caco-2 cancer cell lines, compared with the normal Vero cells. Cell-cycle arrest, apoptosis, and expression of apoptosis-related genes were assessed on PC-3 cells. Both extracts had significant antiproliferative effects on all tested cell lines in a dose-dependent manner, with the mesocarp extract being more potent. Both extracts also showed high antibacterial and antifungal activities. These results were related to the chemical constituents of the extracts identified by the GC–MS analysis. The extract of neem fruit mesocarp caused cell-cycle arrest at G2/M phase of PC-3 cells. The cytotoxicity of neem mesocarp extract is strongly correlated with the induction of apoptosis, where it caused downregulation of the antiapoptotic BCL2 gene but upregulation of the proapoptotic P53 and BAX genes. This study showed that neem fruit extract is potential anticancer material in the future. Although cancer is considered one of the most common devastating diseases and is ranked as the second leading cause of death in humans, currently available cancer chemotherapeutic agents are not tissue-specific. This results in adverse effects on the host cells, thereby increasing morbidity. Disadvantages of monotargeted therapies including ineffectiveness, lack of safety, and excessive cost have encouraged many pharmaceutical companies to invest in the research of multitargeted therapies. Natural plant-based products provide solutions for all these disadvantages being multitargeting, nontoxic or less toxic, and readily available at low cost compared to synthetic agents ,2,3,4. TAzadirachta indica A. Juss. For over 2000 years, neem has been used in traditional medicine in India to treat a range of ailments propyl ester, - in neem fruit mesocarp and epicarp extracts, respectively. Desulfosinigrin is a hydrolysis product of glucosinolates, which have been proven to play a role in reducing cancer risk. The inhibitory activity of desulfosinigrin was observed toward cyclin-dependent kinase by Krishnaveni , upon whg et al.  revealedKB, where it suppressed the antiapoptotic genes (BCL2), whereas it induced the expression of the apoptotic genes  [The neem fruit mesocarp extract was characterized by the presence of d-Glucose, 4-O-à-D-glucopyranosyl- ; octadecanoic acid, 2,3-dihydroxypropyl ester; 1,2-benzenedicarboxylic acid, bis(2-ethylhexyl) ester; hexadecanoic acid 2,3-dihydroxypropyl ester; and 12-Octadecadienoic acid -. Most of these have been reported as potent bioactive components. The spectrum of antibacterial activity was ascribed by several authors to the presence of hexadecanoic acid, 2,3-dihydroxypropyl ester  and 1,2-ed PARP) . Other fed PARP) . Staphylococcus aureus (Gram-positive) than Vibrio cholerae (Gram-negative) and anticancer activity against the normal blood lymphocytes, and the breast cancer cell line (MDA-MB-231). Natarajan et al. [To develop a new anticancer drug, it is of high importance to inspect the regulatory mechanism of cell growth and apoptosis cells as an essential cell-death process, which is considered a potential pathway for the development of new drugs ,39. It in et al.  showed hThe methanolic extracts of neem fruit mesocarp and epicarp had significant antiproliferative effects on all tested cell lines  in a dose-dependent manner, with the mesocarp extract being more potent against the three cell lines. Both extracts also showed high antibacterial and antifungal activities. The GC–MS analysis of the two neem fruit extracts showed the presence of many phytochemicals with evident biological activities, which could be related to their antimicrobial and antiproliferative activities. The methanolic extract of neem fruit mesocarp caused cell-cycle arrest at G2/M phase of treated PC-3 cells. The cytotoxicity of neem mesocarp extract is strongly correlated with the induction of apoptosis, where it caused downregulation of the antiapoptotic BCL2 gene but upregulation of the proapoptotic P53 and BAX genes.


Bacillus subtilis was developed as a chassis organism for engineered endosymbionts (EES) that escape phagosome destruction, reside in the cytoplasm of mammalian cells, and secrete proteins that are transported to the nucleus to impact host cell response and function. Two synthetic operons encoding either the mammalian transcription factors Stat-1 and Klf6 or Klf4 and Gata-3 were recombined into the genome of B. subtilis expressing listeriolysin O (LLO) from Listeria monocytogenes and expressed from regulated promoters. Controlled expression of the mammalian proteins from B. subtilis LLO in the cytoplasm of J774A.1 macrophage/monocyte cells altered surface marker, cytokine and chemokine expression. Modulation of host cell fates displayed some expected patterns towards anti- or pro-inflammatory phenotypes by each of the distinct transcription factor pairs with further demonstration of complex regulation caused by a combination of the EES interaction and transcription factors. Expressing mammalian transcription factors from engineered intracellular B. subtilis as engineered endosymbionts comprises a new tool for directing host cell gene expression for therapeutic and research purposes.Developing modular tools that direct mammalian cell function and activity through controlled delivery of essential regulators would improve methods of guiding tissue regeneration, enhancing cellular-based therapeutics and modulating immune responses. To address this challenge,  The establishment of non-pathogenic engineered endosymbionts through B. subtilis is presented, with the aim of delivering mammalian transcription factors to the host cell for therapeutics and research. This process can be mimicked in the laboratory, as was demonstrated by engineering Escherichia coli to survive within cells of Saccharomyces cerevisiae2. In addition, bacteria can be engineered to express mammalian transcription factors (TFs) that alter cell fate. Extracellular Pseudomonas aeruginosa that deliver TFs via type III secretion into induced pluripotent stem cells (iPSCs) can direct the differentiation of the iPSCs into cardiomyocytes3. Together, these studies support the development of non-pathogenic engineered endosymbionts (EES) that persist in the host cell cytoplasm and influence control of mammalian gene expression. The term EES refers to the functional combination of bacteria that remain viable in the cytoplasm of mammalian cells with the engineered production of modulators  that can redirect host cell biology.The endosymbiont theory of the origin of eukaryotic cells postulates that there was a close and long-term biological interaction (symbiosis) between separate single-cell organisms that led to the genesis of organelles4 provide a useful model. Because phagocytic immune cells readily internalize bacteria and demonstrate altered cell fate that is the result of specific TFs, these cells represent a testable system for EES function4. The immune response is delicately balanced in mammalian systems, both recognizing self and building tissues and defending against disease and foreign invaders capable of damage. Macrophages are commonly recruited by stimuli in response to inflammation and contribute to progression or suppression of associated pathologies5, thus representing a significant component of the inflammatory microenvironment6. Macrophages may prove to be a key cell for molecular therapies directed at modifying cellular functions, since these cells are present within injured, damaged and malignant tissues and can be modulated to switch phenotypes to alter the disease course7. Macrophages are plastic, with the ability to alternate between synthesizing pro-inflammatory or anti-inflammatory signals8 and their function is influenced by the microenvironment in which they reside. Pro-inflammatory macrophages (M1) act in a fashion to destroy pathogens, and anti-inflammatory (M2) macrophages decrease inflammation, support angiogenesis and promote tissue remodeling and repair10. This dichotomy is known as macrophage polarization. Polarized inflammation effector states are distinguished by changes in cell surface markers including, cluster of differentiation (CD)86 for M1 or CD206 for M2, or through differential expression of cytokines and chemokines11.To demonstrate both cytoplasmic persistence of the EES and host cell fate alteration, phagocytic immune cellsBacillus subtilis expressing listeriolysin O (LLO) from Listeria monocytogenes is an engineered intracellular bacterium13. LLO lyses the phagocytic vacuole, releasing internalized bacteria into the cytosol. The hlyA gene encoding LLO was placed under control of an isopropyl β-D-1-thiogalactopyranoside- (IPTG) inducible promoter and inserted into the genome of B. subtilis13. Since B. subtilis expressing LLO can access the cytoplasm and does not have a lipopolysaccharide- (LPS) mediated immune response14, it was chosen as a chassis organism for the development of a cell fate-controlling EES. Additionally, B. subtilis is a non-pathogenic, Gram-positive, soil bacterium that respires as a facultative anaerobe making it capable of replicating in the host cell cytoplasm15. This bacterium has been classically used for secreting complex proteins into the surrounding extracellular space through the general secretory (Sec) and twin-arginine translocation (Tat) pathways16. B. subtilis has been well characterized to the point of full genome annotation and databases (e.g. BsubCyc database) have been developed for metabolism analysis and protein production17. Several sugar-regulated inducible systems18 including D-mannose, which has been shown to be actively transported inside of mammalian cells, provide additional techniques to regulate EES gene expression20. B. subtilis is an ideal chassis organism for development of an EES.Mycobacterium bovis strain used as a tuberculosis vaccine) bacteriotherapy has become standard of care for bladder cancer, and other clinical applications for bacteriotherapy are being tested27. Here, the EES can be used to modulate mammalian cell function by expressing engineered operons that encode mammalian TFs that are delivered to the nuclei of mammalian cells  and Krüppel-like factor 6 (KLF6) which induce a general response to an inflammatory state in macrophages, and the second encodes Krüppel-like factor 4 (KLF4) and GATA binding protein 3 (GATA-3) which are both characterized to drive an anti-inflammatory response in macrophages37. Both STAT-1 and KLF4 are upstream regulators that impact several pathways, while KLF6 and GATA-3 are more specific in regulation, which lends to a dual approach towards driving the desired cell fates37. When expressed from intracellular EES these TFs altered patterns of cell surface markers, cytokine and chemokine expression with some patterns of modulation towards anti- or pro-inflammatory phenotypes, indicating that the EES may be used to direct immune cell function and elucidate mechanisms of macrophage response to intracellular bacteria.Bacteria have been developed that impact mammalian cell physiology for therapeutic approaches, bacteriotherapy, and advances in this field support the development of EES for cellular control. Bacille Calmette-Guerin  localization was compared to LAMP-138 positive structures  in J774A.1 cells (green) with and without IPTG induction of LLO expression (+IPTG and -IPTG). The LAMP-1 protein is crucial for phagosomal assembly and therefore will reveal when the LLO strain is contained within the phagosomes and when the phagosomes have been disrupted39. When LLO expression was induced by IPTG, many of the LLO strain were intact and present throughout the mammalian cells  in the cytoplasm, live cell imaging was performed to image the interaction of the fluorescently stained LLO strain in live J774A.1 cells. With IPTG induction, the LLO strain was observed to replicate in the host cell cytoplasm after phagosomal escape indicating active metabolism and viability  at 2 different time points. At 1 h (h) post-bacterial addition, there was significant change in host cell viability only at the highest MOI  (strain designated LLO-lacZ) through the twin-arginine translocation (Tat) pathway by synthesizing the PhoD signal peptide40 and the amino acids from 126-132 of the simian virus (SV) 40 nuclear localization signal (NLS)41. The production of β-gal by LLO-lacZ, with and without a nuclear localization signal , was studied with IPTG or mannose control. The mannose-inducible system was amplified from the genome of the B. subtilis strain 16819 to provide a genetic switch specifically for controlling protein delivery to nucleus. The mannose-inducible system was chosen due to the characterized uptake of this sugar in mammalian cells20. Localization of the reporter protein to J774A.1 nuclei was confirmed after co-incubation with LLO-lacZ and end-point fluorescence microscopy , the nuclear to background fluorescence signal to noise ratio  and cells exposed to the LLO-lacZ-no NLS strain  or KLF4 and GATA-3   and KLF4/GATA-3 (IL-4 and IL-13) produced some increase in nuclear SNR but significantly less than the LLO-SK and LLO-KG strains over the 3 h.β-gal delivery by the LLO-B. subtilis LLO strain TF delivery on J774A.1 modulation, surface marker expression was examined in cells containing various strains  and compared to M0 and M1 + /M2 + polarized J774.1 cells at 24 or 48 h post-incubation. As determined by flow cytometry, engineered B. subtilis LLO strains expressing TFs exhibited different patterns of J774A.1 surface marker expression when compared to the LLO strain , compared to the LLO strain. The same trend was observed at 48 h , at levels comparable to those of the positive control at 24 h (M2+). However, the elevated levels were not sustained as indicated by the 48 h time point. CD86 expression levels were the same in all bacterial conditions at 24 h in comparison to resting cells with no significant differences in comparison to the M1 control  of Listeria monocytogenes has been shown to either not change cytokine and chemokine profiles in a macrophage cell line, or to change profiles of these proteins equally to the wild-type L. monocytogenes in bone marrow derived macrophages44. Therefore, the 168 strain was not used in further comparisons, because the lack of LLO was not expected to impact J774A.1 profiles. LLO-SK and LLO-KG were shown to alter J774A.1 cytokine and chemokine expression patterns relative to the LLO strain as well as positive controls -10, IL-12p40 and tumor necrosis factor alpha (TNF-α)  relative to both the LLO strain and LLO-KG . Several cytokines were only significantly changed at one of the two time points in certain conditions, including IL-6 and macrophage inflammatory protein-2 (MIP-2/CXCL2) Fig. . D-manno38 . Accordingly, this result demonstrated the potential of B. subtilis LLO to impact host cell function within the 3 h timeframe of interaction between the LLO strains and host cells.During immune responses, STAT-1 is part of the pro-inflammatory response and is a potent modulator that is directly upregulated by exposure to pro-inflammatory cytokines such as IFN-γ. This response was shown at 3 h in this study , or the treatment of macrophages with B. subtilis spores can result in either M1 or M2 activation48. Even with these complexities, clear changes were observed that indicate that LLO-SK and LLO-KG impacted surface marker expression in comparison to the LLO strain.In this study macrophage plasticity was modulated using TFs expressed from the EES. Cell surface markers are commonly used to differentiate M1 and M2 polarization206 Fig.  expressi49. These identifying cytokines and chemokines are important when studying disease as they provide information on the function and characteristics of macrophage populations50. The M1 and M2 polarization classifications represent a trend in immune responses but is complex and contains some mixed signals, likely due to bacterial stimulation which can cause these classifications to be incomplete or oversimplified51. Complexities in macrophage activation, phenotype and plasticity were encountered in this study. In some instances, the LLO-SK and LLO-KG strains impacted the host cell as predicted in comparison to the LLO strain alone 56. Therefore, the increase in production of IL-12p40 by LLO-KG is unexpected; however, it is possible GATA-3 could affect IL-12p40 production. The IL-12p40 promoter has a canonical GATA binding site but GATA-3 has not been studied with this promoter56. The LLO-SK strain showed some downregulation of IL-12p40 at 48 h which could be due to STAT-1 driving alternative cytokine production such as IL-27p2857. TNF-α downregulation by LLO-KG is another expected result35 but downregulation by LLO-SK in comparison to the LLO strain indicates further complexity and potential pleiotropic effects of STAT-1. This could be due to TNF-α being regulated by NF-κB60. Furthermore, there may have been metabolic impact on cytokine production due to the addition of D-mannose, which impairs glucose metabolism and is known to suppress the succinate-driven HIF1α activation of IL-1β leading to downregulation of IL-1β62. Accordingly, results indicated that D-mannose had a significant impact on IL-1β and other cytokines and chemokines, which is an important consideration for future EES studies that rely on sugar-inducible systems. Metabolic reprogramming is most likely playing a significant role in response to the chemical inducer of the engineered B. subtilis LLO TF operons and possibly the LLO strain alone62.Although changes in cytokine and chemokine expression vary along the spectrum of macrophage polarization, there are well-documented cytokines which are used to broadly identify M1 or M2 macrophagesone Fig. . The uprSK and LLO-KG strains able to modify expression further in comparison to the LLO strain in some key examples indicating TF specificity. One component of the differential responses were elevated expression of cytokines and chemokines in response to all bacterial strains compared to the positive controls of IFN-γ and LPS (M1+) or IL-4 and IL-13 (M2+). Additionally, the differences in responses to treatment with bacteria compared to the positive controls was larger in the cytokine and chemokine profiles than were observed with the cell surface markers. Enhanced signaling proteins (cytokines and chemokines) response from macrophages, and other professional antigen-presenting cells, compared to cell surface marker expression has been previously observed65. Other factors that could contribute to the observed complex response involves B. subtilis stimulating Toll-like receptor 2 (TLR2) in contrast to IFN-γ and LPS stimulating TLR466. Additionally, viable bacteria can cause a more dynamic response than just bacterial products alone67. Altogether, even with the dynamic responses from the J774A.1 cells in response to complex signals, the LLO-SK and LLO-KG strains were able to change trends in macrophage surface markers, and cytokine/chemokine profiles in comparison to the LLO strain. This observation is further supported by not observing significant differences in J774A.1 viability, percentage of cells containing fluorescently labeled bacteria and distribution of fluorescence intensity (relating to number of bacteria per cell) when comparing the LLO, LLO-SK and LLO-KG strains  is thought to promote tumor growth, metastases and poor outcomes68. TAMs are broadly M2 polarized, and IL-10, IL-12p40 and G-CSF all have been shown to play important roles in impacting the tumor microenvironment through regulation of TAMs70 which could be targeted by the LLO-SK for cancer bacteriotherapy26. Arthritis represents immune polarization where homeostasis is driven to a pro-inflammatory condition71. Here, modulating macrophages towards the M2 phenotype could reduce inflammation in joints. Downregulation of TNF-α and upregulation of IL-10 LLO-KG shows promise for treating damaging inflammatory conditions such as arthritis71. Manipulation of immune cells in vivo has been characterized by low efficacy and lack of innate control72 and the use of EES could circumvent these issues since engineered bacteria can be taken up by phagocytic cells and direct gene expression towards a therapeutic phenotype.Controlling inflammation could be an important biomedical application of the LLO-73. Additionally, chimeric antigen receptor T (CAR-T) cells and CRISPR technologies have proven to be potential futures for some therapeutics and are clinically relevant, but each has some limitations74. Viral vectors have been shown to be slow as therapeutics within the immune system, specifically in targeting and modulating macrophages, compared to exogenous cytokines75. An alternative method of manipulating cellular fates using prosthetic networks increases the variety of cargo that can be delivered and provides some control with limitations73. The EES can build on the precedence of prosthetic networks by having the capability to be constructed to generate complex sets of proteins and molecules once in the cytoplasm of mammalian cells for improved control; continuously supplying TFs, or alternatively providing a method for delivering CRISPR-Cas977, which could improve directing cell fates.The use of an EES is advantageous when compared to alternative methods of manipulating mammalian cell fates and function. Current methods of manipulating mammalian cell fate include viral vectors, growth factors or signaling moleculesB. subtilis serves as an ideal chassis for the development and optimization of the EES capable of surviving in the cytoplasm and delivering proteins to the nuclei of mammalian cells to alter cellular fates.Studies of pathogens and their virulence factors should inform future development of the EES. The EES will utilize this characterization for defined control within the host cell cytoplasm. This study demonstrated the utility of an EES to alter mammalian cell fates. The use of the EES as a tool to change mammalian cell function may have use in the treatment of diseases by altering the function of mammalian cells. B. subtilis LLO at the amyE locus using a homologous recombination plasmid . The pDR111 plasmid was transformed into B. subtilis using a natural competence protocol and constructs were selected by spectinomycin then confirmed by PCR amplification out of the genome79. B. subtilis expressing IPTG-inducible LLO was provided by Dr. Daniel Portnoy. The constructs include the lacZ, Stat-1Klf6 and Klf4Gata-3 genetic cassettes. B. subtilis LLO was designed to secrete β-gal to the nucleus through the twin-arginine translocation (Tat) pathway by synthesizing the PhoD signal peptide40 and the amino acids from 126-132 of the simian virus (SV) 40 nuclear localization signal (NLS)41 together and connecting both to lacZ when inserting into pDR111 using Gibson assembly after lacZ was amplified from the pST5832 plasmid . The same construct was engineered without the SV40 signal to confirm specific delivery to the nucleus. The lacZ gene and the synthesized PhoD signal peptide plus SV40 NLS were cloned into the NheI restriction site in pDR111 using Gibson assembly. Initially, the β-gal secretion strain was controlled by an IPTG-inducible promoter (Phyper-spank). However, previous studies have shown the IPTG system is limited in controlling protein production so these constructs were engineered to be controlled by the mannose-inducible system amplified from B. subtilis ZB307 strain genome19. The mannose promoter and regulator were cloned into the pDR111 plasmid to replace the Phyper-spank promoter and LacI regulator using Gibson assembly. Accordingly, the lacZ gene with the same design was cloned in the NheI restriction site present in the pDR111 mannose plasmid using Gibson assembly. The Stat-1 and Klf6 genes were synthesized by IDT as a custom gene and Gblock, respectively, from the coding sequences obtained from Uniprot. The Stat-1Klf6 operon was fused by ligation at an introduced EagI restriction site between the genes during cloning into the pDR111 mannose plasmid by restriction cloning into the SalI and NheI restriction sites. The Klf4 and Gata-3 genes were synthesized as Gblocks from IDT and fused using the same method as the Stat-1Klf6 operon. The Klf4Gata-3 operon was cloned into the pDR111 mannose plasmid using restriction cloning at the SalI and SbfI restriction sites after the SbfI cut site was introduced into the multiple cloning site by inverse PCR then digesting both ends with SbfI and re-ligating the pDR111 mannose plasmid. All constructs were confirmed by restriction digest, sequencing and functionality tests. A list of primers, Gblocks and custom genes are included in Supplementary Data Constructs were inserted into the genome of B. subtilis LLO was grown under the same conditions for all experiments. Each B. subtilis LLO construct was grown in Luria-Bertani Miller broth (LB) with the appropriate antibiotic. B. subtilis LLO was grown in LB with chloramphenicol (10 µg/mL) and all constructs that were integrated into the amyE were grown with spectinomycin (100 µg/mL). The overnight cultures were grown for 16 h at 37 °C and 250 RPM. All constructs were integrated into the genome of B. subtilis LLO which allowed for expression of constructs without antibiotics during co-incubation with J774A.1 cells. B. subtilis 168  was grown in LB with no antibiotic selection overnight for 16 h at 37 °C and 250 RPM.B. subtilis LLO delivery, unless otherwise described. J774A.1 monocyte/macrophage cells  were maintained at 37 °C and 5% CO2 in DMEM , supplemented with 10% fetal bovine serum (FBS). Cells were tested negative for mycoplasma using the MycoAlert PLUS Mycoplasma Detection Kit . Once cells were confluent, they were seeded onto a plate or 4-well chambered imaging slide and allowed to adhere overnight (described below) when an estimation of total number of cells was made based on confluency. B. subtilis LLO was added at an optimized MOI of 25:1 for all experiments besides testing of host cell viability, along with IPTG (500 µM) to induce expression of LLO with or without protein of interest. B. subtilis LLO and J774A.1 cells were then co-incubated at 37 °C and 5% CO2 for 1 h J774A.1 cells were then washed three times with PBS and new medium was added containing gentamicin (4 µM) to eliminate any remaining extracellular B. subtilis LLO. Co-incubation continued for 3 h at 37 °C and 5% CO2 prior to imaging or preparation for microscopy (described below)  at 4 °C overnight followed by a goat anti-rabbit IgG Dylight 650  secondary antibody at room temperature (RT) for 2 h. Phagosome formation or destruction was shown by incubating an anti-Lamp-138 antibody  at 4 °C overnight followed by a goat anti-rat IgG Alexa Fluor 555  secondary antibody at RT for 2 h. Nuclei were counterstained by incubating cells with Hoechst 33342 (1 µg/mL) for 10 min (min) at RT. Membranes of the cells were stained by incubating cells with PKH67 green-fluorescent cell linker kit  for 10 min at room temperature. Slides were then coverslipped using Fluoromount-D mounting media . Slides were imaged using confocal microscopy (described below).After fixation with 4% paraformaldehyde (PFA), cells were permeabilized using 0.3% Triton X-100 (ThermoFisher) followed by a blocking step containing 0.3% Triton X-100 and 5% normal goat serum . B. subtilis LLO escape from the phagosome complex by imaging J774A.1 cells that had been treated with the B. subtilis LLO with and without IPTG. Imaging was performed using a 60x oil objective and 1.5x zoom and using filter sets for DAPI (Hoechst 33342), GFP (PKH67), TRITC (Alexa Fluor 555 for LAMP-1) and Cy5 (Dylight 650 for B. subtilis). Z-stacks were taken at 0.5 µm steps to confirm location of B. subtilis LLO within host cells. Images were analyzed using NIS-Elements AR Software (Nikon) and background noise was reduced by using Nikon denoise.ai algorithm. The three-dimensional volume images and cutaways were produced by the Alpha display mode. The Alpha display mode was also used to generate three-dimensional videos to display z-depth location of the B. subtilis LLO during escape from the phagosomes.Confocal microscopy was performed using a Nikon A1 CLSM  microscope to determine B. subtilis LLO on J774A.1 cell viability was determined after EES delivery. Conditions examined were: multiple time points of interaction between B. subtilis LLO and host cells , different MOIs , no IPTG induction and no treatment, with biological triplicates (n = 3) for each time and condition. At each time point, J774A.1 cells were washed once with PBS then MTS reagent (Abcam) was added to cells at a 10-fold dilution with DMEM followed by incubation for 30 min at 37 °C then absorbance was measured at 490 nm. All treatment conditions were compared to J774A.1 cells alone to elucidate any differences in loss of viability of the J774A.1 cells due to the treatment conditions. Flow cytometry was used to analyze cell viability after engineered B. subtilis delivery at 10:1, 25:1 and 50:1, compared to no IPTG induction and no treatment at the 4 h time point (n = 1) or after the addition of LLO, LLO-SK and LLO-KG  compared to no treatment. Furthermore, uptake of B. subtilis LLO, LLO-SK and LLO-KG by J774A.1 cells was assessed after staining bacteria with CellTracker Orange CMRA Dye . Following staining, the strains were washed three times before adding to J774A.1 cells at 25:1 MOI for 4 h incubation. Cells were collected, washed once with 1X PBS and incubated with Zombie NIR viability dye  in PBS for 20 min, at 4 °C in the dark. Cells were washed twice followed by fixation using 4% PFA and resuspended in 100 µL flow buffer for analysis using the Cytek Aurora Cytometer . All samples were assessed for percent live cells. Cells which were incubated with CTO bacteria were assessed for percent CTO positive cells (indicating J774A.1 cells containing bacteria) and CTO MFI was used as a relative measure of CTO bacteria per cell. Standard one-way ANOVA with Tukey post-hoc test was used to determine statistically different values.The effect of B. subtilis LLO was internalized into J774A.1 cells as described above, using a 96-well black glass-bottom plate . B. subtilis LLO was stained with CellTracker Orange CMRA Dye  as described above then added to J774A.1 cells. Live cell imaging was performed on a Leica DMi8 Thunder microscope equipped with a DFC9000 GTC sCMOS camera and LAS-X software . Cells were maintained at 37 °C and 5% CO2 in Fluorobrite medium during the imaging session. Fluorescent images of CTO were acquired using a TRITC filter set. Brightfield and fluorescent images were acquired consecutively, using a 63x oil objective every 1 h beginning at 1 h post co-incubation and continuing until 4 h post-incubation. Z-stacks were taken at all time points at 0.4 µm steps to confirm B. subtilis LLO presence within cytoplasm. B. subtilis LLO presence in J774A.1 cells was quantified using Fiji (ImageJ) software and cell counter plugin by counting >1.5 µm rods throughout z-depth. An area of 2090 µm by 1254 µm in each well was imaged and used to perform this quantification.B. subtilis LLO engineered to secrete β-gal (LLO-lacZ) with and without a NLS (LLO-lacZ-NLS and LLO-lacZ-no NLS) were internalized into J774A.1 cells as described above, using 4-well chambered slides . Incubation was also performed using the supernatant from a 16 h mannose-induced LLO-lacZ-NLS culture. The second incubation step was 3 h and began after adding gentamicin (4 µg/mL) and with and without D-mannose (1% w/v) for the β-gal secretions strains. During extended studies, the cells were washed gently 3 times with 1X PBS then a high concentration of gentamicin (25 µg/mL) was added after 3 h and incubation continued for an additional 21 h. The cells were then fixed with 4% PFA for 10 min prior to preparation for microscopy (described below).E. coli) antibody-rabbit  at 4 °C overnight followed by a goat anti-rabbit IgG Dylight 650  secondary antibody at RT for 2 h Nuclei were counterstained by incubating cells with Hoechst 33342 (1 µg/mL) for 10 min at room temperature. Slides were then coverslipped using Fluoromount-D mounting media . Epi-fluorescent microscopy was performed using a Nikon Eclipse Ci-L microscope equipped with a CoolSNAP DYNO camera for fluorescent imaging and NIS elements BR 5.21.02 software (Nikon). Images were acquired with a 40x phase contrast objective and for fluorescent imaging DAPI and Cy5 filter sets were used. Nuclear SNR was quantified by imaging in random areas in each corner of the well and in the center of the well. At least 5 random images totaling an area of 1085 µm by 825 µm were utilized in drawing regions of interest (ROIs) around nuclei of J774A.1 cells to quantify Cy5 fluorescence by utilizing Hoechst 33342 counterstain to determine nuclei location. Nuclear SNR was calculated by using mean fluorescence intensity of ROI region around nuclei of J774A.1 cells in each of the five random images  divided by standard deviation (SD) of noise in the well. SD of noise in well was determined by drawing 3 ROIs (550 µm2) in background areas of each image then calculating SD of the mean fluorescence from all ROIs drawn in background areas. Statistics was determined using Brown-Forsythe and Welch ANOVA with Dunnett T3 post-hoc test and all treatment conditions were compared.After fixation with 4% PFA, cells were permeabilized using 0.3% Triton X-100 (ThermoFisher) followed by a blocking step containing 0.3% Triton X-100 and 5% normal goat serum (ThermoFisher). β-gal secretion to the nucleus was identified using fluorescence imaging after incubation with an anti-β-galactosidase , using a 6-well plate (Corning Costar #3516). TFs were secreted for 3 h then allowed to be trafficked for an additional 21 h as described above . For flow cytometry, Accutase  was used to detach J774A.1 cells for analysis (described below). For Luminex cytokine profiling , the supernatant was removed at both 24 and 48 h and then analysis was performed to quantify cytokines produced (described below). Non-stimulated J774A.1 cells were assumed to be at resting state. J774A.1 cells were polarized with interferon-gamma (IFN-γ) and lipopolysaccharide (LPS)  or interleukin (IL)-4 and/or IL-13  to be used as positive controls. Furthermore, J774A.1 cells were treated with the B. subtilis 168 strain in flow cytometry characterization to compare to LLO strain. The EES with operons were treated with and without mannose as described in B. subtilis LLO β-gal secretion and the LLO strain was not as no difference in impact on J774A.1 cells was observed in previous flow experiments. All treatment conditions were performed in biological triplicates (n = 3).The LLO strain, LLO-B. subtilis LLO, LLO-SK and LLO-KG overnight cultures were grown for 16 h at 37 °C and 250 RPM in triplicate (n = 3). All cultures were diluted 1:20 then allowed to grow into logarithmic phase for 3 h Subsequently the cultures were normalized to OD580 = 0.1 then 100 µL was transferred into columns of a 96 well plate  for a total of 24 replicates . Cultures were grown in a PerkinElmer VICTOR Nivo plate reader at 37 °C and 300 RPM with OD580 measurements (580 nm was used due to plate reader limitations) taken every 30 min. Measurements were performed until growth rates began to slow in late logarithmic phase in the 100 µL . All replicates were plotted to visualize differences in growth rates between strains as mean ± SD.B. subtilis LLO strains and controls, followed by incubation for 24 or 48 h, cells were collected and stained in a 96-well round bottom plate. Treatment addition was staggered so that flow staining and data acquisition could be performed at the same time for 24 and 48 h cells. All staining steps were performed in 100 µL volume at 4 °C in the dark. Samples were first incubated with Zombie NIR viability dye  for 20 min. Cells were washed once with flow buffer, followed by incubation with TruStain FcX™ PLUS (anti-mouse CD16/32) Antibody  for 10 min. Alexa Fluor® 647 anti-mouse CD86 Antibody  and FITC anti-mouse CD206 (MMR) antibody  were then added and incubated for 20 min. Cells were washed twice with flow staining buffer and fixed with 4% PFA for 10 min and resuspended in a final volume of 100 µL for flow cytometry analysis using the Cytek Aurora spectral flow cytometer (Cytek). Single stained controls and unstained controls for all conditions were used to assess fluorescent spread and for gating strategies. Flow cytometry data was analyzed with the software FCSExpress . A standard one-way ANOVA with Tukey’s multiple comparisons test was used to determine statistically different MFI values amongst all groups within each time point. A two-way ANOVA with Šidák’s multiple comparisons test was used to compare the 24 and 48 h data for each surface marker. The data presented herein were obtained using instrumentation in the MSU Flow Cytometry Core Facility. The facility is funded in part through the financial support of Michigan State University’s Office of Research & Innovation, College of Osteopathic Medicine, and College of Human Medicine.After addition of the engineered Cell culture supernatant was stored at −20 °C until ready for use. Supernatant was analyzed for CCL2 (MCP-1), CCL3 (MIP-1a), CXCL2/MIP-2, G-CSF, IL-1α, IL-1β, IL-4, IL-6, IL-10, IL-12p40, IL-12p70, IL-13, TNF-α and VEGFα cytokine expression. Cytokine levels of cell supernatants were measured using a MCYTOMAG-70K-17 Mouse Cytokine Magnetic Multiplex Assay (Millipore Sigma) using a Luminex 200 analyzer instrument  according to the manufacturer’s instructions. Standard one-way ANOVA with Tukey post-hoc test was used to determine statistically different values amongst all treatment groups.B. subtilis LLO modulation of J774A.1 cell protein expression was used as the template protocol for this experiment. The experiment was performed using a 96-well black glass-bottom plate . However, J774A.1 cells were fixed at 3 h post mannose addition to reveal engineered B. subtilis LLO delivering TFs to host cells using antibodies against the transcription factor of interest. Each transcription factor was stained for with the appropriate antibody individually within the wells. The strains was stained prior to addition to host cells using Invitrogen CellTracker Orange CMRA Dye (Invitrogen) as in live cell imaging. After fixing, cells were permeabilized using 0.3% Triton X-100 (ThermoFisher) followed by a blocking step containing 0.3% Triton X-100 and 5% normal goat serum (ThermoFisher). Cells were then incubated with rabbit anti-Stat-1 , rabbit anti-Klf6 , rabbit anti-Klf4  or rabbit anti-Gata-3  at 4 °C overnight followed by a goat anti-rabbit IgG Dylight 650  secondary antibody at RT for 2 h Nuclei were stained by incubating cells with Hoechst 33342 (1 µg/mL) for 10 min at room temperature. Membranes of the cells were stained by incubating cells with PKH67 green-fluorescent cell linker kit (10 µM) for 10 min at room temperature. Imaging was performed using Leica DMi8 Thunder microscope equipped with Leica DFC9000 GTC sCMOS camera and Leica LAS-X software (Leica) with the following light emitting diodes for excitation: Hoechst 33342 (395 nm), PKH67 (475 nm), CellTracker Orange CMRA Dye (555 nm) and Dylight 650 for TFs (635 nm) filters were used and imaged using a 40X objective to confirm LLO-SK and LLO-KG delivery of TFs. Images were quantified using Fiji (ImageJ). Thresholding was performed on the Hoechst 33342 images to identify the nuclei. Nuclear ROIs were used to quantify fluorescence intensity in the Dylight 650 channel. Nuclear SNR was calculated using mean nuclear fluorescence/standard deviation of the background signal. Background signal was quantified from one data set for each TF. A mean of 158.4 ± 63.6 (SD) cells were analyzed per data set. Outliers were identified using the ROUT methods (Q = 1%) and outliers removed for analysis using a one-way ANOVA and Tukey’s multiple comparisons test.Protocol for engineered p < .05 was considered a significant finding. All experiments were piloted to optimize the new methods then three independent biological replicates were used unless otherwise specified to examine reproducibility of data. For fluorescent quantification experiments, appropriate representative individuals were quantified using random imaging and quantification methods as to avoid any biases in data acquisition and quantification.Statistical analyses were performed using Prism software . Statistical tests are identified for each method. Plotting was performed using R version 4.0.4 with the following packages: ggplot2, dplyr, reshape2, ggsignif, plotrix and ggpubr. Data are expressed as mean ± standard deviation; Further information on research design is available in the Peer Review FileSupplementary InformationDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Movie 1Supplementary Movie 2Reporting summary