Cancer is the second leading cause of death in the US. Current major treatments for cancer management include surgery, cytotoxic chemotherapy, targeted therapy, radiation therapy, endocrine therapy and immunotherapy. Despite the endeavors and achievements made in treating cancers during the past decades, resistance to classical chemotherapeutic agents and/or novel targeted drugs continues to be a major problem in cancer therapies. Drug resistance, either existing before treatment (intrinsic) or generated after therapy (acquired), is responsible for most relapses of cancer, one of the major causes of death of the disease. Heterogeneity among patients and tumors, and the versatility of cancer to circumvent therapies make drug resistance more challenging to deal with. Better understanding the mechanisms of drug resistance is required to provide guidance to future cancer treatment and achieve better outcomes. In this review, intrinsic and acquired resistance will be discussed. In addition, new discoveries in mechanisms of drug resistance will be reviewed. Particularly, we will highlight roles of ATP in drug resistance by discussing recent findings of exceptionally high levels of intratumoral extracellular ATP as well as intracellular ATP internalized from extracellular environment. The complexity of drug resistance development suggests that combinational and personalized therapies, which should take ATP into consideration, might provide better strategies and improved efficacy for fighting drug resistance in cancer. In 2017, about 1.7 million people were diagnosed with cancer and 0.6 million people died from the disease. Drug resistance and the resulting ineffectiveness of the drug treatment are responsible for up to 90% of the cancer related deaths.Cancer is the second leading cause of death in the USDrug resistance in cancer is a well-known phenomenon that results when cancer becomes tolerant to pharmaceutical treatment. Resistance to anticancer drugs arises from a wide variety of factors, such as genetic mutations and/or epigenetic changes, conserved but upregulated drug efflux, and various other cellular and molecular mechanisms.. Despite the achievements made in treating cancers during the past decades, resistance to classical chemotherapeutic agents and/or novel targeted drugs continue to be a major problem in cancer therapies and responsible for most relapses, one of the major causes of death in cancer. Many classical chemotherapeutic anticancer agents kill cancer cells by directly damaging their DNA, which has the problem of non-specificity and relatively high toxicity. In recent decades, more and more targeted drugs have been developed to precisely target/block changes that drive cancer growth and proliferation. Although these drugs show remarkable effects during initial treatment, a large majority of patients develop resistance as treatment proceeds. For example, 30%-55% of patients with non-small cell lung cancer (NSCLC) relapse and die from the disease afterwards. The 50%-70% of ovarian adenocarcinomas reoccur within 1 year after surgery and associated chemotherapy. About 20% of pediatric acute lymphoblastic leukemia patients develop recurrence.Current major treatments for cancer management include surgery, cytotoxic chemotherapy, targeted therapy, radiation therapy, endocrine therapy and immunotherapyBetter understanding of mechanisms underlying the acquisition of drug resistance is urgently needed and will facilitate developing novel therapeutic strategies and lead to better clinical outcomes. In this review, we will describe intrinsic and acquired drug resistance, present new discoveries in specific mechanisms of drug resistance and discuss new strategies for fighting against drug resistance and improving anticancer efficacy.,17.Drug resistance can be categorized as intrinsic or acquired resistance based on the time when it is developed. Intrinsic resistance exists before drug treatment while the acquired resistance is induced after therapy, each occurring in about 50% of cancer patients with drug resistanceIntrinsic resistance is usually defined as the innate resistance that exists before the patient is administered with (exposed to) drugs, which usually causes reduced efficacy of the drug treatment. Intrinsic resistance can be caused by: (1) pre-existing (inherent) genetic mutations in a majority of tumors that result in decreased responsiveness of cancer cells, such as triple negative breast cancer cells, to both chemo and target drugs; (2) heterogeneity of tumors in which pre-existed insensitive subpopulations, including cancer stem cells, will be selected upon drug treatment thus leading to relapse in later stages of therapeutic treatment; (3) activation of intrinsic pathways used as defense against environmental toxins (such as anticancer drugs).. The high expression of HER2 gene promoted the upregulation of transcription factor Snail, inducing a morphological change similar to epithelial-mesenchymal transition (EMT), which renders cancer cells more resistant. Furthermore, the HER2/Snail double positive patients were found to have lower survival rate than patients with only single positive or double negative genetic makeup.Intrinsic drug resistance may exist in cancer cells prior to therapies due to the presence of genetic mutation(s) of genes involved in cancer cell growth and/or apoptosis. For example, poorer outcome of cisplatin treatment in gastric cancer patients was found to be associated with HER2 overexpression. The latter two activities make cancer stem cells (CSCs) more resistant to radiation and chemotherapies. It was also shown that resistant cells are more mesenchymal-like,21. These concomitant changes link intrinsic drug resistance with EMT and CSCs.It was shown that transcriptional repressors Snail and Slug not only mediate EMT, but also resistance to p53 induced apoptosis, and a self-renewal program. Patients would respond to therapy initially because the majority of the tumor cells are sensitive to the drug. However, the resistant subclones would proliferate after the drug treatment and cause recurrence. This intrinsic drug resistance is sometimes mistaken for acquired resistance since the tumor would shrink first upon treatment and the resistance seems to be acquired due to therapy. CSCs are a subpopulation in tumors with the capacity of self-renewal and differentiation, participating in tumor initiation and progression. They have been reported to be involved in resistance to chemotherapeutic drugs in multiple cancer types, including leukemia, glioblastoma and pancreatic cancer. Combined therapy targeting both CSCs and most tumor cells might be required to reduce drug resistance.Pre-existing resistant subpopulations in tumors may also lead to relapse after chemotherapeutic treatment. Increasing evidence shows that existence of intratumoral genetic heterogeneity in primary tumors predates clinical intervention and glutathione (GSH)/glutathione S-transferase system, working to reduce cellular drug accumulation or detoxify drug treated cancer cells respectively.Therapeutic effects of a drug can be reduced by activation of intrinsic pathways that are used as a defense against environmental toxins, including anticancer drugs. Examples of these protective mechanisms include ATP binding cassette (ABC) transporter mediated drug effluxAcquired resistance can be identified by gradual reduction of anticancer efficacy of a drug after the drug treatment. Acquired resistance can be a result of: (1) activation of second proto-oncogene that becomes the newly emerged driver gene; (2) mutations or altered expression levels of the drug targets; (3) changes in tumor microenvironment (TME) after treatment.. The comparison of mutations between primary and relapse tumors revealed novel gene mutations. In addition, the results showed increased transversion mutations in relapsed tumors, suggesting that cytotoxic chemotherapeutic drugs caused DNA damage in cancer cells and might increase probability of the emergence of new mutations.Shrunk tumors can acquire resistance and regrow due to new mutations. In one study, genomic profiles before and after relapse of eight acute myeloid leukemia patients were analyzed using whole-genome sequencing. One mechanism of the resistance is due to a point mutation of T315I of the fusion tyrosine kinase protein. The change of threonine 315 to isoleucine results in a loss of a hydrogen bond, which is necessary for the binding of imatinib to the ATP-binding site of BCR-ABL, leading to significantly reduced efficacy of the drug.Cancer cells can acquire resistance to targeted drugs when the genes encoding target proteins develop new mutations or change expression levels. One example of secondary mutations within the target kinase is the threonine 315 to isoleucine (T315I) mutation in the BCR-ABL kinase domain. Tyrosine kinase inhibitor (TKI) imatinib targeting BCR-ABL is commonly used in chronic myelogenous leukemia treatment, but approximately 20%~30% of patients will experience resistance or relapse after treatment. In cisplatin treated neuroblastoma (NBL) tumor, NBL cells secret exosomic miR-21 to induce TAMs to produce exosomic miR-155 to in turn silence the TERF1 gene in NBL cells. TERF1 protein is an inhibitor of telomerase, and decreased expression of TERF1 would result in increased telomerase activity and resistance to chemotherapy. Therefore, the exchange of exosomic miRNAs between tumor cells and stromal cells in TME can promote drug resistance.Drug resistance can also be acquired due to the dynamic changes of TME in the course of treatment. During malignancy progression and development of resistance, cross-talk exists between tumor cells and their microenvironment. Exosomes released by cancer and stromal cells are involved in the crosstalk. Researchers found that exosomes, which are released by cancer cells and carry certain miRNAs were used by cancer cells and tumor-associated macrophages (TAMs) in the TME to communicate with each otherMechanisms of intrinsic and acquired resistance described above can co-exist during tumor progression and treatment. Mechanisms of acquired drug resistance can be totally different from the pre-existing intrinsic drug resistance. Alternatively, it can be a selective expansion of the intrinsic drug resistance.The degree of intrinsic drug resistance pre-determines the sensitivity of given cancer cells to a specific drug. Genomic and other biochemical analyses should be performed before the drug treatment plan is designed to avoid potential pre-existing drug resistance. After acquired drug resistance is developed, therapeutic schemes need to be adjusted accordingly.One of the objectives of drug treatment should be to effectively slow down or stop tumor growth without inducing acquired, or at least uncontrollable acquired drug resistance. The best drug treatment plan should take prevention or delay of acquired drug resistance into consideration.Although it is scientifically important to distinguish intrinsic and acquired resistances, the specific mechanisms of resistance are more clinically significant.,39,40. The resistance caused by the abnormally high rates of drug efflux could be either intrinsic or acquired, depending on if it exists prior to or develops after drug administration.Elevated efflux of anticancer agents, which leads to decreased intracellular drug accumulation, has been considered to be the major reason for chemotherapy resistanceABC genes and they are classified into seven subfamilies (ABCA-ABCG),42. Among them, ABCB1, ABCC1 and ABCG2 are highly involved in the acquisition of multidrug resistance (MDR) to cancer chemotherapeutics.Transmembrane transporters responsible for the drug efflux are primarily from the ABC transporter superfamily. The human genome contains 48 . ABCB1 has multiple drug binding sites that can bind and pump a wide variety of substrates from the cell, such as etoposide, doxorubicin, paclitaxel and vinblastine. High expression level of ABCB1 has been observed before chemotherapy in many different tumor types, including kidney, lung, liver, colon and rectum. In contrast, initially low expression and then dramatic increased expression of ABCB1 post chemotherapy were observed in many hematological malignancies, such as AML and ALL.ABCB1 (MDR1 or P-gp) is one of the most well-characterized ABC transporters. It is composed of two transmembrane domains that form a passage for substrates and two nucleotide-binding domains that bind and hydrolyze ATP. The binding and the subsequent hydrolysis of ATP is coupled with conformational changes in the transporter, leading to the pumping out of the transport substrates. While ABCB1 transports amphipathic and lipid-soluble compounds, ABCC1 pumps organic anionic substrates such as compounds conjugated to glutathione, glucuronide, or sulfate. Overexpression of ABCC1 has been shown to be associated with resistance in many cancer types including lung, breast and prostate cancers,57,58.ABCC1, or multidrug resistance-associated protein 1 (MRP1), similar to ABCB1, is also responsible for pumping out a wide variety of anticancer agents, such as vinca alkaloids, anthracyclines, epipodophyllotoxins, camptothecins, and methotrexate,59,60. Besides breast cancer, ABCG2 overexpression was also found in many other cancer types including lung cancer and leukemia,61.ABCG2, or breast cancer resistance protein, is the major drug efflux transporter in breast cancer associated resistance, as indicated by the name. ABCG2 is considered a marker of CSCs in some cancers, and responsible for the side-population effect. It can transport both positively- or negatively-charged drugs, ranging from chemotherapeutic drugs  to several TKIs (imatinib and gefitinib). For example, ABCC2 and ABCC3 can transport many chemotherapeutic drugs, including cisplatin, doxorubicin, and etoposide, and their overexpression results in multidrug resistance. Mutations and overexpression of ABC transporters directly influence tumor sensitivity and drugs’ anticancer efficacy. An accurate and complete expression profile of ABC transporters in tumors is important for proper drug selection and better treatment outcomes.Other ABC transporters have also been studied for their substrates and functions in tumor resistance to anticancer drugs, providing additional explanations on the mechanisms of drug resistanceCompared to traditional chemotherapies which kill cancer cells by disrupting rapid cell proliferation and may affect normal dividing cells, targeted therapies can block the growth of cancer cells by inhibiting the activity of specific target proteins involved in tumor development, thus being more selective and effective to cancer cells and less harmful to normal cells. However, targeted therapy may also develop the problem of resistance, resulting from alteration of drug targets. The alteration of drug targets may be either a secondary mutation in the target protein or changes in expression levels due to epigenetic alterations.,67. However, almost 50% of the responsive patients would develop a T790M mutation on EGFR within one year, resulting in resistance to the first and second generations of TKIs. The mutation from threonine to methionine led to a configuration change in EGFR and consequently enhanced ATP binding affinity and impaired binding of gefitinib/erlotinib for the kinase,71. To overcome the resistance caused by T790M, third generation of TKIs, like osimertinib and rociletinib, has been developed and reported to show clinical efficacy with patients harboring T790M mutation,73. However, resistance to third-generation inhibitors develops not long after their use, raising the need of developing fourth generation TKIs. One reported major mechanism of the new resistance is due to a mutation in EGFR known as C797S. The loss of the cysteine residue, which is important for TKIs to target the ATP site, impairs the binding of the third generation TKIs to EGFR. Therefore, EAI045, a fourth generation TKI targeting both T790M and C797S, has been designed to bind an allosteric site located on EGFR, attempting to circumvent the mechanism patterns of resistance to the early generations of TKIs which all bind to the ATP sites,76. The battle between generation of new genetic mutations and generation of new TKIs that restore drug sensitivity may become a new trend in the everlasting war against drug resistance.For example, TKIs of the epidermal growth factor receptor (EGFR), such as erlotinib and gefitinib targeting NSCLC, have been reported to show high response rate at initial treatment,78. Given the problems with TAM and the quest for alternative drugs, aromatase inhibitors (AIs) were developed, working by interfering with the last step of estrogen synthesis. Third-generation AIs are now being used as first-line therapy in postmenopausal women with hormone receptor positive breast cancer.The development and use of estrogen receptor inhibitors in breast cancer treatment provide another example for the resistance induced by alteration of the drug target. Tamoxifen (TAM) is commonly used for patients with ER-positive breast cancer, relying on its ability to compete with estrogen for the ligand binding site of ER. However, extended exposure of TAM often leads to drug resistance. Mechanisms of resistance vary in different cases, and mutations in the ER gene and decrease in ER expression level are among them. For example, genes involved in DNA repair, like FEN1, FANCG, RAD23B, were found to be upregulated in 5-FU resistant human colon cancer cell lines,82. 5-FU treatment induced upregulation of p53-target genes on DNA damage response and repair. Success to repair the damages led to reduced cell cycle arrest and apoptosis in the resistant cell lines compared to parental cell lines.Many chemotherapy drugs, like cisplatin and 5-fluorouracil (5-FU), kill cancer cells by inducing DNA damage. The DNA damage response (DDR) of affected cells to the anti-cancer drugs may result in reduced efficacy of the drugs by DNA lesion repairs, leading to drug resistanceAlthough deregulation of DDR may remit the resistance induced by DNA repair, it may also increase the risk of developing new mutations due to genomic instability, the accumulation of which may initiate a new round of carcinogenesis. Therefore, DNA damage response is a complex mechanism in cancer treatment and recurrence, and it requires thorough consideration when used as an anticancer therapeutic target.INK4a. Cellular senescence can be triggered by endogenous and exogenous stimuli, among which the three major stimuli include excessive mitogenic signaling produced by activated oncogenes, telomere shortening, and non-telomeric DNA damage caused by chemotherapeutic drugs. For example, Doxorubicin and Cisplatin used in chemotherapy by inducing cell death can also initiate senescence,86.Cellular senescence refers to irreversible arrest of cell proliferation, largely leading to activation of tumor suppressive pathways mediated by p53 and/or p16. Cancer cells with TIS can gain stem-cell properties, which accounts for the escape from senescence and cancer relapse,89.Escape from therapy-induced senescence (TIS) has been recognized as a mechanism for drug resistance and tumor recurrence/progressionAn emerging mechanism contributing to drug resistance is epigenetic alterations. Increasing evidence brought people’s attention to epigenetic modifications that also participate in the development of other mechanisms of resistance, including increased drug efflux, enhanced DNA repair, and impaired apoptosis.. Overexpression of Tβ4 led to the acquisition of stem cell-like capacity in the HCC cell line and induced resistance to VEGFR inhibitor sorafenib in vivo.Epigenetic modifications include DNA methylation, histone modification, chromatin remodeling, and non-coding RNA related alterations. For example, demethylation of DNA at the promoter region of an oncogene would upregulate the expression of the gene, resulting in drug resistance. A recent study demonstrated that a G-actin monomer binding protein thymosin β4 (Tβ4) was enriched through demethylation of DNA and active modification of histone H3 at the promoter region in a resistant hepatocellular carcinoma (HCC) cell line,97. MiRNAs contain about 21-25 nucleotides (nt) while lncRNAs can range from 200 to more than 10,000 nt in length. MiRNAs are known to be important regulators of post-transcriptional gene expression by binding to their complementary mRNAs and mediating mRNA degradation and repression of protein synthesis. LncRNAs participate in gene expression regulation in different ways such as blocking the binding of transcription activators to key DNA sequences in genes or recruiting chromatin remodeling proteins. Both miRNA and lncRNA regulate expression of proteins related to cancer drug resistance. For example, lncRNA urothelial cancer-associated 1 (UCA1) was shown to be upregulated in cisplatin-resistant bladder cancer cells compared to sensitive cells. Upregulation of UCA1 expression resulted in significantly increased mRNA and protein levels of wingless-type MMTV integration site family member 6 (Wnt6), promoting Wnt signaling and cell survival.Besides chromosomal modification, non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), also play an important role in drug resistanceetc.), metabolic heterogeneity in oxygen/nutrient distribution, and temporal heterogeneity in dynamic tumor progression. Tumor heterogeneity increases the complexity and difficulty of cancer treatment, making it almost impossible to kill all cancer cells using one single therapeutic. This problem led to the development of combinational therapies used in many cancer treatments such as FEC: 5-FU, epirubicin, cyclophosphamide for breast cancer. In this section, genetic heterogeneity will be the focus of discussion.Four levels of heterogeneity are present in tumors: genetic heterogeneity, cell type heterogeneity  that work together to contribute to all aspects of the hallmarks of cancervs. pHi6.8-7.2). In contrast, cancer cells develop a so-called “reversed pH gradient”, with increased intracellular pH and decreased extracellular pH, through the proton pumping of proton transporters and the modulation of pH sensors,110. The acidic (pH6.5-7.1) extracellular environment of cancer cells has been reported to be a contributor to the resistance to chemotherapeutics. The reversed pH gradient impairs the distribution of weak base anticancer drugs, which is described as “ion trapping”, and enables cancer cells to evade apoptosis,113. As an emerging hallmark of solid tumors, the low extracellular pH would be a potential target for cancer therapy. Therapeutic approaches attempting to reduce microenvironment acidity, such as proton pump inhibitors (PPIs), have been developed and shown good efficacy in shrinking tumor and sensitizing cancer cells to chemotherapy drugs. Lansoprazole, an example of PPI, has been reported to demonstrate synergistic effects when used in combination with paclitaxel in melanoma cells both in vitro and in vivo.TME may contribute to intrinsic resistance to anticancer therapies. One of the TME factors is pH. In normal tissues and cells, extracellular pH is usually slightly higher than intracellular pH (pHe7.3-7.5 . Macrophages secrete high levels of colony stimulating factor-1 (CSF-1) in GBM tumors, supporting cancer cell proliferation and survival,116. Therefore, CSF-1 receptor (CSF-1R) has been targeted by small molecule inhibitors or antibodies in cancer treatment with promising in vivo effects. However, more than 50% GBM patients suffer from recurrence, which is derived from elevated secretion of insulin-like growth factor-1 (IGF-1) from TAMs and IGF-1 induced elevation of phosphatidylinositol 3-kinase (PI3K) pathway signaling in GBM tumor cells. Combining inhibition of CSF-1R with inhibition of IGF-1 receptor or PI3K signaling has been reported to extend overall survival in mouse models. Thereby, combined therapies simultaneously targeting cancer cells and TME may produce much improved anticancer efficacy by reducing drug resistance.Changes in the composition of TME after treatment also contribute to the adaptation of cancer cells to chemo or targeted therapeutics, thereby reducing drug efficacy and inducing resistance. For example, TAMs play a role in the acquisition of resistance in response to anticancer therapies in glioblastoma multiforme (GBM), a severe type of brain tumor. The frequent cycles of hypoxia and reoxygenation produce oxidative stress that could induce DNA damages in tumor cells, thus contributing to genetic instability that leads to accumulation of additional mutations and emergence of genetically divergent clonal subpopulations. Moreover, as mentioned previously, cells in TME, like TAMs, interfere with the expression profiles of cancer cells by releasing miRNA-containing exosomes, thus contributing to tumor heterogeneity.Besides TME heterogeneity per se being one aspect of tumor heterogeneity, it also contributes to the enrichment of genetic heterogeneity. For example, due to variation and dynamic nature of vasculature inside tumors, fluctuating hypoxia is one characteristic of TMETherefore, TME plays very significant role in tumor progression and therapeutic resistance. Better understanding and targeting/manipulating TME and its interaction with tumor cells could substantially enhance therapy response and achieve better clinical outcomes.et al. reported that EMT promotes resistance to apoptosis induction activated by drug cyclophosphamide in an EMT lineage-tracing mouse system. However, the mechanisms of EMT-induced drug resistance are not fully understood although most recent studies suggest that EMT and CSC share some similarities and their involvements in drug resistance represent different manifestations of the same phenotype. One possible mechanism is that EMT cells share many similarities in signaling pathways with cancer stem cells (CSCs), such as Wnt, Notch and Hedgehog pathways. Therefore, EMT enables tumor cells to gain resistance to anticancer drugs and evade drug induced cell death. For example, TGF-β is a well-studied key cytokine in EMT, the signaling pathways of which are correlated with gain of drug resistance,125. Inhibition of TGF-β can reverse the process of EMT and remarkably increased the sensitivity of cancer cells to chemotherapies,127. Wnt and Hedgehog pathways are also reported to be related with drug resistance,129.EMT is a process during which epithelial cells lose their attachment to each other and gain the characteristics of mesenchymal stem cells. EMT has been known to be essential for the initiation of metastasis in tumors of epithelial origin, but it is not so clear about its role in other tumors such as sarcomas. Increasing evidence shows that EMT plays a critical role in chemotherapy resistance. Fischer .Furthermore, accumulating evidence shows that the CSCs rely on the EMT program as a critical regulator when mediating drug resistance. The CSC state of carcinoma cells requires epigenetic changes resulted from activation of EMT. Understanding the mechanistic linkage between EMT, CSC and drug resistance would significantly contribute to anticancer therapeutics. One recent study reported that suppressing EMT by knocking out EMT transcription factors Twist1 or Snail1 enhanced sensitivity to gemcitabine and increased survival rate in pancreatic ductal adenocarcinoma-bearing mice treated with the drug. Some of these EMT-TFs promote resistance by enhancing drug efflux by ABC transporters. Promoters of genes coding for ABC transporters were found to have EMT-TF binding sites. Overexpression of Twist, ZEB1/2, Slug, and Snail enhances expression and activity of ABCB1, thus inducing drug resistance. ABCG2, another ABC transporter closely linked to MDR, is known to be regulated by Snail, MSX2, SOX2 and ZEB1. Other ABC transporters involved in MDR, such as ABCC1, ABCC2, ABCC4, and ABCC5, also have been shown under the regulation of EMT-TFs. For example, overexpression of ABCC5 is correlated with FOXM1 in paclitaxel-resistant nasopharyngeal carcinoma cells. Depletion of either FOXM1 or ABCC5 decreases drug efflux and increases cell death induced by paclitaxel. Knockdown of these EMT-TFs sensitize cancer cells to chemotherapeutic drugs by suppressing ABC transporters,143,148. Targeting these EMT-TFs may inhibit metastasis and drug resistance at the same time.EMT inducing transcriptional factors (EMT-TFs) also play roles in promoting drug resistance. Overexpression of EMT-TFs like Twist, Snail, Slug, ZEB and FOXC2 are known to induce drug resistance. MiRNAs are small endogenous RNA of 20-24 nucleotides, which have a complex network and can regulate expression of genes that are associated with EMT and are of ABC family. Haenisch et al. have summarized miRNA-mediated regulations of ABC transporters. MiRNA network regulates ABC transporters at different levels, with the majority of miRNAs acting at post-transcriptional levels via binding sites at the three prime untranslated region (3’-UTR) and some miRNAs acting at transcriptional levels by binding to the gene promoter region. miRNAs can not only regulate expression of ABC transporters but also regulate EMT markers. For example, miR-200c is reported to both down-regulate multiple ABC transporters including ABCB1 and ABCG2,153, and negatively regulate EMT by directly targeting 3’-UTR regions of ZEB1 and ZEB2, and retain the epithelial phenotype. Some miRNAs, such as miR-200c and miR-145, inhibit ABC transporters and suppress EMT; other miRNAs like miR-27a positively regulate ABC transporters and promote EMT,157.Besides EMT-TFs, miRNAs are believed to be essential molecules that link EMT and ABC transporterset al.. One recent study revealed that over expression of miR-134, miR-487b, and miR-655 promotes TGF-β induced EMT and drug resistance to gefitinib in NSCLC. This miRNA cluster induces resistance to EGFR-TKI by directly inhibiting MAGI2, leading to reduced PTEN activity. Reduction of PTEN and upregulation of PI3K-Akt-pathway is associated with acquired EGFR-TKI resistance.MiRNAs also regulate drug resistance by other mechanisms including regulating apoptosis and autophagy, controlling anti-cancer drug metabolism, modulating drug targets and DNA repair, and regulating GSH biosynthesis, which has been summarized by An All these discussed drug resistance mechanisms are schematically shown in Drug resistance can be intrinsic or extrinsic in nature. Drug resistance is induced at levels of DNA , RNA (microRNA), and proteins [concentration and activity changes of receptor tyrosine kinases ]. In addition, different stromal cells and stressed/lysed cancer cells in a tumor release ATP into intratumoral space, creating a high ATP concentration TME. Intratumoral extracellular ATP (eATP) works as a messenger outside of cancer cells through purinergic signaling cascade to induce EMT, which contributes to drug resistance. eATP is also internalized by cancer cells via macropinocytosis, leading to greatly elevated intracellular ATP (iATP) levels. The higher iATP levels in turn enhance the efflux activity of ABC transporters for pumping anticancer drugs out of cancer cells, increase competition between iATP with ATP analog anticancer drugs at the intracellular ATP binding domain of RTKs located on cancer cell plasma membrane and increase RTK phosphorylation. All these mechanisms work together dependent or independent of ATP to augment drug resistance by reducing intracellular drug concentration, increasing cell survival signaling and inducing EMT (cancer stem cell-like features). More studies are needed for the final validation of ATP-mediated mechanisms of drug resistance.. Furthermore, even higher intracellular ATP levels were found in acquired resistant cancer cells lines compared to their parental cell lines,169. In one study using colon cancer cells lines, intracellular ATP levels were found to have a two-fold increase in the chemo-resistant cell lines compared to those in their drug-sensitive parental cell lines. The authors demonstrated that ATP levels played a pivotal role in multiple drug resistance by showing that artificial delivery of ATP into the drug-sensitive cells resulted in drug resistance while depleting intracellular ATP with a glycolysis inhibitor sensitized resistant cancer cells. A study from another group also showed that intracellular ATP contributed to drug resistance to cisplatin in ovarian adenocarcinoma cells. In contrast to the more glycolysis-related ATP elevation in the previous study, the authors showed that the resistant cell line had an increased intracellular ATP level as a consequence of enhanced mitochondrial ATP synthesis that was induced during the development of resistance. They suggested that the resistant cell line may have increased their energy storage for the purpose of protecting the cells from xenobiotics and environmental stress. These studies showed the metabolic- (energetic-) role of intracellular ATP in acquired resistance to chemotherapeutic drugs. Elevated intracellular ATP levels is likely to be a necessary condition for cancer cells particularly resistant cancer cells.ATP, either intracellular or extracellular, plays significant roles in cancer cell growth, survival and resistance. It is known that the intracellular ATP level in cancer cells is higher than that in normal tissues of the same origin, likely due to the upregulated glucose transport and aerobic glycolysis in cancer cells, a process also called the Warburg effect.For all the reasons mentioned above, inhibitors targeting glycolysis related proteins (GAPDH and LDH) like 3-bromopyruvate, oxamate and FX11 that significantly deplete intracellular ATP levels might sensitize cancer cells to anticancer drugs3 to 104 times higher than those in their corresponding normal tissues. The functions of this high eATP have been under investigation. In our recent study, eight anticancer drugs, including both targeted and chemotherapeutic drugs, were tested in five cancer cell lines of five different organ origins, and extracellular ATP was found to promote intracellular ATP increase and cancer cell survival in most cases. When studying ATP promoted drug resistance to sunitinib in NSCLC A549 cells, we found that extracellular ATP can be internalized by cancer cells through macropinocytosis and other endocytic mechanisms, resulting in substantially elevated intracellular ATP levels from 150 to 200% of the original intracellular ATP concentrations,179,180. Thus, macropinocytosis, and other endocytosis-mediated extracellular ATP internalization, and the resulting intracellular ATP level elevation is responsible, at least in part, for the observed drug resistance.eATP levels of various cancer types have been reported to be 10, which suggested the dual roles of ATP as both an energy molecule facilitating drug efflux and signal-transduction (phosphorylation) molecule activating cell survival signaling pathways.One of the possible drug resistance mechanisms induced by the internalization of extracellular ATP is that more abundant intracellular ATP molecules compete with tyrosine kinase inhibitors which are ATP competitors, for the ATP binding site located on RTKs, leading to increased phosphorylation and activation of downstream signaling pathways. Meanwhile, the internalized ATP molecules also enhance efflux of TKIs and chemo drugs by ABC transporters, resulting in decreased drug accumulation and increased cell survival. The reduced intracellular drug concentrations and elevated intracellular ATP levels further enhance the ATP binding and reduce TKI binding on RTKs, leading to even more RTK-mediated signaling and drug resistance. These were all observed in the study. Extracellular ATP was also shown to alter expression levels of ABC transporters, indicating the profound effects of ATP on modulating ABC transporter activities at both transporting activity rate and transporter expression levels to potentially efflux anticancer drugs and enhance drug resistance. Prescreening of tumors based on their ability of ATP internalization (macropinocytosis) and expression of specific set of ABC transporters will provide valuable information for the selection of proper anticancer drugs and prediction of patient’s response to therapeutics, therefore reducing drug resistance and enhancing the efficacy of drug treatment. Our lab’s findings in extracellular ATP’s roles in cancer cell growth, survival and drug resistance were reviewed in a 2018 Nature Reviews Cancer paper.In the five cell lines studied, increased intracellular ATP was found to correlate with drug resistance status when the ABC transporters expressed by the cell line matched those required for the efflux of a given drugThe drug resistance observed here is intrinsic in nature and could last during later treatments. Reducing intratumoral extracellular ATP concentration and/or blocking ATP internalization may increase drug efficacy at both initial and later stages of cancer treatment.,183. Purinergic receptors such as members of the P2X and P2Y families were reported to be involved in cancer drug resistance. In one recent study, researchers demonstrated that ATP promoted resistance to chemotherapeutic drugs in colorectal cancer cells through P2Y-mediated upregulation of MRP2 and concurrent drug pumping. Another study showed that P2X7 receptor, when activated by ATP, exhibited anti-apoptotic activity in methoxyestradiol-treated melanoma cells. Extracellular ATP has also been found to upregulate expression of glucose transporter 1, possibly via the P2X7 induced PI3K-AKT pathway and hypoxia-inducible factor 1α-dependent signaling. These changes are also likely to increase cancer cell survival and drug resistance. Therefore, reducing the extracellular ATP concentration could increase drug efficacy by reducing specific purinergic receptor signaling in addition to reducing ATP internalization. An ATPase, apyrase, was shown to reduce the growth of glioblastoma when injected in a rat glioma model, suggesting the feasibility of this strategy.ATP might also contribute to cancer drug resistance from outside of the cell through purinergic receptor signaling. Purinergic signaling has been known to promote cell growth and proliferation,188-191. Defects in the molecular machinery for autophagy in cancer cells or purinergic receptors in immune cells lead to poor response to stimuli that would cause immunogenically induced cell death,192. Furthermore, immune cells in TME can release and use extracellular ATP, potentially creating an ATP-rich and tumor-friendly environment. For example, lymphocytes are known to release large amounts of ATP as a signaling molecule into the extracellular space when stimulated, serving as a messenger in cellular interactions of T lymphocytes. Stimulated monocytes can release ATP as an autocrine signal molecule. ATP released from these immune cells as well as dying or stressed cells can be sequentially converted to AMP and adenosine by cell surface enzymes CD39  and CD73 ,196. Adenosine mediated signaling would lead to the establishment of an immunosuppressive environment,198. Combined administration of an inhibitor of extracellular ATPase and a synthetic TLR4 ligand was reported to restore the infiltration of necroptosis-deficient tumors by APCs and CD8+ T cells, and re-establish normal sensitivity to mitoxantrone-based chemotherapy. These findings suggest that extracellular ATP is essential to anti-tumor immune response while its hydrolysis products contribute to immunosuppression. Therefore, the anti-resistance strategy through ATP degradation by ATPase is perhaps not appropriate to use together with therapies designed to boost immune system. However, this may be an extracellular ATP concentration-dependent phenomenon. There will be a concentration window for extracellular ATP where extracellular ATP concentrations are sufficiently low not to generate significant drug resistance but sufficiently high to maintain anti-tumor immune responses. Significantly more studies need to be done before we fully understand the different functions and different concentrations of extracellular ATP on cancer and immune cells in tumors.Extracellular ATP also plays significant role in immunoregulation in TME and potentially affects therapeutic results. ATP can be secreted into extracellular environment by cancer cells undergoing autophagy or apoptosis induced by chemotherapy or radiotherapy, generating a chemotactic gradient around dying cells and recruiting myeloid cells through purinergic signalingDue to the high heterogeneity among tumors growing in patients and high complexity of evolution of tumor progression, identifying the best strategy to overcome drug resistance will be very challenging. On the other hand, with the development of high throughput cancer genomics, cancer proteomics, and cancer metabolomics analyses, it is now possible to identify driver genes and major components that contribute the drug resistance the most at any specific stage of tumorigenesis in a patient. Due to the nature of individual differences in mutations in multiple and different cancer-causing genes, combinational and personalized therapies are required. Combinational therapies are strongly preferred since tumors are almost always multi-clonal and genetically heterogeneous. Therapeutic strategies using single drugs are most likely to lead to eventual treatment failure due to drug resistance as the treatment kills sensitive cancer cells but leaves resistant cancer cells to survive and proliferate. In comparison, combinational therapy using two or more drugs is likely to target multiple driver genes simultaneously, not only inhibiting more clones in a tumor but also making new cancer mutations resistant to multi-drug treatment much more difficult to be selected and grow up.Current strategies to deal with drug resistance depend on continuous monitoring of patients and treatment with a cocktail of chemotherapeutic/target drugs, each targeting one or more proteins encoded by driver genes responsible for drug resistance pathways operating in cancer patients. Like recent trends of successful target drug therapy, simultaneous multi-targeting will be more effective in combating drug resistance, thereby enhancing anticancer efficacy of therapies and prolong patients’ lives. However, the outcomes of therapies inevitably depend on the composition/unique resistance profile of tumors and the toxicity tolerance of patients, so that the therapeutic results are hard to predict. Since tumor cells could always develop alternative mechanisms to circumvent current therapy, fighting against drug resistance seems to be an endless game.. In one study, researchers found that melanoma cells that acquired resistance to combined BRAF- and MEK- targeted therapy displayed robust drug addiction and were exquisitely sensitive to acute drug withdrawal. This drug sensitivity is also detected in other cancer types such as lymphoma cells treated with an ALK kinase inhibitor that became dependent on it, suggesting that intermittent dosing may prolong control of ALK+ tumors. These findings encouraged testing of this pulsatile dosing regimen in clinical trials .Conventionally, cancers were treated with chemo or targeted drugs at the highest dosage that the patients can tolerate. In recent years, it was realized that such a treatment strategy may lead to drug resistance more rapidly, since such treatment puts constant pressure on tumors to select those cancer cells that are strongly resistant to the drugs. New treatment strategies of “on and off” or “high dose followed by low dose” resulted in longer survival and delayed drug resistance, because this intermittent or adaptive dosing may interrupt the growth of drug-dependent resistant cells and allow the competition of sensitive and resistant cellsHowever, these new treatment strategies produced inconsistent results in that improved survival was achieved in some cases but no change or even worse outcomes occurred in others. These indicate that the new strategies only work in some specific cases but cannot be used as a general strategy. We still need to know more about drug-cancer interactions at a personalized level before we can develop treatment plans that fit individual needs.,204. Some cancer cells strongly prefer to use glucose as the energy and carbon sources. These cancer cells are “addicted” to glucose and are more sensitive to glucose concentration changes than normal cells, dying significantly faster than normal cells under glucose deprivation,167,205. In those cancer cases, using a glucose transport inhibitor or a glycolysis inhibitor in combination with another target drug may be particularly effective in inducing cancer cell death.One possible strategy of circumventing resistance is to block energy supply of tumor cells. Tumor might bypass any mechanism, but could not evade the need for energy to support their growth, proliferation and other activities such as drug resistance and cell migration. Combinational therapy with an addition of blockage of energy may increase efficacy of therapeutic reagents. Although normal cells are more versatile and flexible in using various energy supply molecules, cancer cells appear to be more rigid in using energy sources. For example, it is well known from PET-scans that aggressive tumors with poor prognosis are almost always glucose/energy metabolism active tumors,179,181. Although different molecules can be used as the energy source for synthesizing ATP, the ultimate energy molecule used in all cells is ATP. Cancer cells appear to have to have higher ATP levels for survival and drug resistance. This difference between cancer and normal cells can be explored to combat cancer growth. If a method, such as tumor-selective targeting, can be developed and used to deliver ATP synthesis-inhibitor or extracellular ATP degrader resulting in reduction of ATP internalization inside tumors, tumors will be deficient in ATP, forcing them to stop growing or even undergo cell death. Extracellular ATP degradation or the inhibition of extracellular ATP internalization can be considered for combinational therapy with chemotherapeutics to enhance the anticancer efficacy of TKIs and chemo drugs. It is also conceivable that alteration of extracellular ATP levels may further enhance cancer immunotherapy, as immune cells in tumors are sensitive to ATP levels.New cancer research indicates that TME plays very important roles in tumorigenesis and drug resistance. Any new therapies that expect to significantly improve the therapeutic outcomes must consider TME - tumor interactions. Intratumoral extracellular ATP has emerged as one of TME molecules that exert profound impacts on tumor cells regarding cell growth, survival, drug resistance and even metastasisFinally, the earlier the tumor is detected, the lower the heterogeneity of tumor cells would be, and the less drug resistance and the more successful the therapy should be. Early prevention and early detection should be considered with at least equal importance as cancer treatment at advanced stages.


Biological therapy is considered an alternative treatment capable of eliciting the same effects on tumors as surgery, radiotherapy, and chemotherapy. As a major player in biological therapy, oncolytic viruses (OVs) have attracted great attention and achieved good results. Specifically, the successful application of OVs in head and neck cancer, as well as melanoma, promoted its research in triple negative breast cancer (TNBC). TNBC is a high-risk molecular type of breast cancer, characterized by strong invasion, easy recurrence, and metastasis. Due to the absence of estrogen and progesterone receptors, as well as the absence of overexpression or gene amplification of human epidermal growth factor receptor 2 (HER2), endocrine therapy and anti HER-2 targeted therapy have proven ineffective. Although chemotherapy has shown substantial efficacy in some TNBC patients, the occurrence of drug resistance and poor prognosis have prompted the exploration of new and effective treatment methods. The emerging concept of OVs provides a new platform to treat TNBC. Indeed, several studies have confirmed the therapeutic effects of OVs in TNBC. Numerous studies have also investigated the efficacy of OVs in other malignances, including solid tumor clinical trials, thus further demonstrating the promising application of oncolytic virotherapy for TNBC. The primary focus of the current review is the examination of OV mechanisms underlying their antitumor properties, while also summarizing the ongoing progress in OV research regarding TNBC treatment, as well as the various combinatorial strategies comprising OVs and other therapies. We also briefly introduce specific relevant clinical trials and discuss some of the progress in the research of novel OVs for the treatment of other malignancies, thereby affirming the significant therapeutic potential of OVs for the treatment of TNBC, as well as other cancers. Oncolytic virotherapy is a new biological treatment method under extensive research. Oncolytic viruses (OVs) are natural or artificially modified viruses that selectively infect and destroy tumor cells, rarely damaging normal cells. The tendency of OVs to infect tumor cells is attributed to the specific cytokines expressed by malignant cells and damaged signal pathways that lead to the destruction of cellular antiviral defense, thereby making tumor cells particularly susceptible to OVs , 2. OVs Breast cancer has a high prevalence rate and the highest mortality rate in women . Breast Currently, a few OVs have been approved for clinical application. Oncorine (H101), a recombinant oncolytic adenovirus was approved for the treatment of head and neck cancer by the China Food and Drug Administration in 2005 . TalimogInitially, the understanding of the antitumor effect of OVs was limited. They were considered to replicate and directly lyse the tumor cells after infecting them, a process termed oncolysis. However, further research revealed that the antitumor effect of OVs is the result of multiple mechanisms Fig. , which aTumor growth requires a large supply of nutrients and oxygen as well as the excretory system for excreting metabolites generated due to the infinite proliferative character of tumor cells. Host vasculature is inadequate to meet these demands; thus, angiogenesis is initiated. Considering the role of angiogenesis in tumors, its inhibitors, such as Bevacizumab, were developed for cancer treatment. OVs were revealed to play an antitumor role by affecting tumor vessels. Studies revealed that some OVs destroy tumor vasculature via the infection and direct lysis of tumor-associated endothelial cells (ECs) as well as lead tumor cells to inflammatory responses and release of tumor necrosis factor (TNF)-α and interferon (IFN)-γ . In addiAnother remarkable antitumor mechanism of OVs is their induction of immune response. OV infection recruits innate immune cells, such as natural killer (NK) cells and neutrophils that form a defense system for virus elimination. Meanwhile, some of these cells also produce an antitumor effect. For instance, NK cells kill malignant cells by releasing cytotoxic granules, such as perforin and granzymes, as well as by secreting death receptor ligands (FasL and TNF-related apoptosis inducing ligand (TRAIL)) and cytokines, such as TNF-α, granulocyte-macrophage-colony-stimulating factor (GM-CSF), and IFN-γ , 28. AddIn the past 5 years, the novel immune-based concept of ‘hot’ and ‘cold’ tumors has been proposed. Tumors are classified based on the distribution of immune cells within the tumor site by determining immune cell infiltration and Immunoscore ranges from I0 (for low immune cell densities with the absence of both cell types in both regions) to I4 (high immune cell densities in both locations). Tumors with non-infiltrates corresponding to Immunoscore I0 are designated as ‘cold’ tumors, while highly infiltrated tumors corresponding to Immunoscore I4 are called ‘hot’ tumors . Due to Viruses can carry foreign genes, replicate in the host, and are readily modified in vitro. As such, viruses are often used as vectors for genetic engineering, the rapid development of which has facilitated the application of OVs for the induction of antitumor effect by arming them with target genes that either encode antitumor cytokines or promote antitumor immune responses , 38, 39.In summary, OVs represent potential candidates for the treatment of cancers owing to their abilities to replicate in tumor cells causing their direct lysis, destroy tumor vasculature, activate innate and adaptive immune responses to generate an antitumor effect, converting ‘cold’ tumors to ‘hot’ ones, and function as a vector, carrying target genes and expressing antitumor related factors in the tumor cells. These characteristics and antitumor mechanisms make OVs advantageous compared to other treatment methods, including radiotherapy, chemotherapy, and endocrine therapy. Moreover, their preference for tumor cells makes OVs highly effective, with fewer adverse events and drug resistance. Further, since viruses actively replicate themselves after entering host cells, causing subsequent activation of the immune system, oncolytic virotherapy is also capable of maintaining long-term antitumor effects.TNBC, a high-risk breast cancer subtype, has limited treatment options and, thus, may benefit from oncolytic virotherapy. Indeed, several studies on the application of oncolytic virotherapy in TNBC have demonstrated the feasibility of this treatment strategy. Here, we introduce different OVs that have been studied in TNBC. In addition, we summarize OVs that have reportedly achieved good results in preclinical studies Table  41–60].–60.41–60Adenoviruses dsDNA viruses are the most studied OVs in breast cancer. Several studies have employed insertion of genes that express antitumor or immune regulatory cytokines in OVs to enhance their effects. IL-24 is an important immune mediator and a cancer suppressor that can specifically inhibit the growth of tumor cells and induce cancer cell apoptosis , 62. ZhuHSV, another dsDNA OV, is one of the only two OV agents approved for clinical application. Nevertheless, researchers continue to study its application in the treatment of other tumors, including breast cancer. G47Δ, an oncolytic HSV with the US11 promoter region, and deletion of α47, inhibits tumor growth and increases survival rate in vitro in human breast cancer MDA-MB-435 cells, as well as in vivo in a mouse model generated by the implantation of MDA-MB-435 cells or tumor xenografts . AdditioVaccinia virus is a unique dsDNA virus that can replicate in the cell cytoplasm. As an OV, vaccinia virus has great prospective application in the treatment of breast cancer. As IL-24-armed adenovirus exhibited a marked antitumor effect, the vaccinia virus strain Guang9 (VG9) was designed as a vector for carrying IL-24 gene. This recombined virus (VG9-IL-24) kills infected breast cancer cell lines without having any marked cytotoxic effect on normal cells. Next, its antitumor effect was evaluated in vivo by establishing an MDA-MB-231 xenograft mouse model. The mice treated with VG9-IL-24 showed slower tumor progression and longer survival periods with a higher survival rate . As MDA-As a dsRNA OV, reovirus has the potential to be used as a new treatment for breast cancer. A study found that several breast cancer cell lines are susceptible to reovirus infection, while normal breast epithelial cells did not express such characteristic, regardless of hormone receptor status . Moreove+ T cells, VSV exerted a significant therapeutic effect. This effect on the immune response suggested that VSV in combination with checkpoint inhibitors has the potential to treat TNBC [VSV is a nonpathogenic negative-sense ssRNA virus. Its characteristic sensitivity to the antiviral activity of type-1 IFNs in normal cells but insensitivity to tumor cells ensures its rapid replication in tumor cells . Symptomeat TNBC .As early as 2006, reports suggested the potential of MV to treat breast cancer. A study reported that infection with an MV strain, which produces carcinoembryonic antigen (MV-CEA), resulted in MDA-MB-231 cell death in vitro. Further, they tested the antitumor ability of MV-CEA in vivo by establishing subcutaneous MDA-MB-231 xenografts, and concluded that MV-CEA had potent therapeutic efficacy against TNBC both in vitro and in vivo . FurtherNDV belongs to negative-sense ssRNA virus group. The AF2240 strain is one of the most effective oncolytic NDVs that is capable of stimulating apoptosis in breast cancer cells , 84. In Maraba virus is a new OVs used in virotherapy and has also been used to treat breast cancer. To verify the possibility of neoadjuvant oncolytic virotherapy, a research team used single direct intratumoral injection of Maraba virus to patient-derived xenografts (PDXs) of TNBC. They concluded that the injection of Maraba virus into 4 T1 tumors effectively initiated virus infection and killing of tumor cells. In subsequent experiments, they developed a rechallenge model to force the reemergence of the disease. They concluded that neoadjuvant oncolytic virotherapy using Maraba virus generated a relatively persistent personalized immune response to kill tumor cells . This stCoxsackievirus is an enterovirus with positive-sense ssRNA and has 29 subtypes. Because of its slight pathogenicity to humans, the oncolytic effect of this virus was relatively less studied. However, in upcoming years, genetic engineering can enhance the selectivity of the viruses to tumor cells and reduce their pathogenicity, making the use of coxsackievirus as an OV possible. Coxsackievirus B3 (CV-B3) was genetically engineered to inhibit many tumor cell types, including lung cancer and endometrial cancer –89. CoxsIn addition to the OVs mentioned above, many others are currently being investigated for their clinical efficacy. Although their successful application for the treatment of TNBC remains to be confirmed, the findings thus far in other malignant tumors suggest that they represent additional potential candidates for TNBC treatment. For instance, poliovirus, which can invade the central nervous system and damage the motor neurons, was found to possess antitumor properties, and suppress the growth of breast cancer in vivo and in vitro using xenografts . The antDue to the heterogeneity of cancer, the effect of monotherapy is often unsatisfactory, making combination therapy undoubtedly the best choice for cancer treatment. Meanwhile, the therapeutic potential of OVs can further promote the progress of combination therapy. The effect of such combination therapies have been demonstrated in other cancers as well. For instance, Mori et al. reported a case where a patient with platinum resistant ovarian cancer, with a long history of treatment who was frequently refractory to traditional therapies, showed significant clinical response to combined gl-onc1 OV therapy and chemotherapy . CurrentAs radiation may induce killing of OVs, research on the combination of OVs and radiotherapy is limited. Nevertheless, the synergistic antitumor effect of OVs combined with radiotherapy has been confirmed. For instance, Dai et al. observed that oncolytic HSV synergistically enhanced cytolysis in pancreatic cancer cells with radiation via apoptosis, to achieve a therapeutic effect . MoreoveIn addition to chemotherapy and radiotherapy, immune therapy as a new method of cancer treatment has been proposed in recent years. Immune checkpoint (IC) inhibitors (ICIs) showed good therapeutic effects in a variety of cancers –115. OneIn recent years, both OVs and chimeric antigen receptor T cell (CAR-T) therapy have received extensive attention. CAR-T cells are genetically engineered to express specific receptors that recognize specific surface antigens expressed on cell surfaces . As suchAdditionally, by modifying T cells with chimeric antigen receptors that recognize antigens on the surface of cancer cells, CAR-T cells can be designed to specifically target cancer cells . HoweverAlthough modification of OVs, in the studies described here, greatly reduced their toxicity compared with that of wild type viruses, it remains necessary to assess the toxicity of these viruses. An important reason for the use of OVs in cancer treatment is their preference for infecting tumor cells. However, this does not mean that OVs pose zero threat to normal cells. Considering that cancer patients generally receive chemotherapy, radiotherapy, and other treatments that cause immune impairment, there is an inherent risk of serious adverse events developing from OV therapy. Another challenge is the directed delivery of OVs to the tumor site, which is the most critical step in its clinical application. Intravenous infusion is undoubtedly the most convenient and feasible method for drug administration, however, this mode is unsuitable for OV delivery, as they elicit their antitumor effects only after entering tumor cells. Although selective toward tumor cells, OVs are recognized as xenogeneic, thereby inducing immune responses leading to interactions with antigen-presenting cells and resulting in the release of proinflammatory factors . Hence, Coating viral particles with polyethylene glycol, which may prevent their interaction with blood cells, thus, increasing their circulation time in blood stream, was found to reduce transduction of hepatocytes and hepatotoxicity, thereby increasing the anticancer efficacy after systemic (intravenous) administration of OV . Other sFuture research must focus on the development of an effective method to reduce the elimination of intravenously infused OVs, thus ensuring that they reach the tumors and replicate. This is essential as the antiviral immunity activated by OVs also suppress the efficacy of oncolytic virotherapy. Furthermore, the timing of OV application has not been thoroughly investigated. Currently, considering that there are no alternatives to surgical interventions, it is necessary to determine if the oncolysis treatment is most efficacious when performed before or after surgery. This is especially important since the replication of OVs requires tumor cells. Thus, OVs may lack a sufficient amount of replication sites post resection of solid tumors, resulting in decreased therapeutic efficiency.Based on previous clinical data, combination therapy should be the first choice for treatment strategies. Hence, additional data on combination therapies specific for TNBC are needed. Moreover, further clarification of the underlying mechanisms associated with the synergistic effect of combinatorial therapy comprising OVs and radiotherapy or chemotherapy, is required. Moreover, combined treatment need not be limited to two methods. Interestingly, a clinical trial combined the OV, pelareorep, PD-1 inhibitor pembrolizumab, and a chemotherapeutic agent did not induce significant toxicity while showing encouraging efficacy for the treatment of patients with advanced pancreatic adenocarcinoma in this phase Ib study . It is, Although OVs have shown promising effects in TNBC treatment, based on in vitro and in vivo models, clinical and experimental evidence remains sparce. Nevertheless, clinical trials performed for OVs in other tumors portend the feasibility of their application for TNBC . Hence, OVs can be considered potential candidates for the treatment of TNBC owing to their abilities to replicate in tumor cells, causing their direct lysis, destroy tumor vasculature, activate innate and adaptive immune responses to generate an antitumor effect, transform ‘cold’ tumors to ‘hot’, act as a vector carrying target genes and expressing antitumor related factors in tumor cells. These characteristics make OVs advantageous, compared with other treatment modalities, including radiotherapy, chemotherapy, and endocrine therapy. Moreover, their preference for tumor cells reduces the adverse events elicited by OVs to normal cells, while also reducing the development of drug resistance. Moreover, as the viruses actively replicate following entry into host cells, they effectively activate the immune system, thus, maintaining a long-term antitumor effect. Hence, to address the urgent requirement for advanced and novel treatment strategies for TNBC, the feasibility of OVs must continue to be investigated as a means of opening new avenues of research and providing a novel platform for clinical treatment.


Objective: This study aims to explore the factors influencing long-term-care beneficiaries’ decisions between home health care and institutional care in South Korea. This study further investigated the association between these two types of long-term care services and emotional, financial, and physical burden alleviation among the beneficiaries and their family members. Methods: We analyzed data from 2019 Long-Term Care Survey. Out of 5,606 respondents, 4,079 long-term care beneficiaries and family members were included in this study. Logistic regression models were conducted to understand factors associated with types of long-term care services, and the association between types of long-term care services and burden alleviation. Results: Having a spouse, having children, or a shorter geographic distance between the beneficiaries and their family members were factors associated with higher likelihood of receiving home health care rather than institutional care. Also, more sever mobility limitation, outpatient care, and fall experiences were associated with higher likelihood of receiving institutional care. Utilization of institutional care was significantly associated with higher alleviation of physical burden. No significant differences between home health care and institutional care were found on emotional and financial burden alleviation. Conclusions: The findings suggest that family resources may be beneficial for older adults to pursue aging in place in later life. However, the physical toll of their family members may exist. Our findings provide evidence to inform public policy decisions on long-term care services for older adults and their family members.


The metazoan body plan is established during early embryogenesis via collective cell rearrangements and evolutionarily conserved gene networks, as part of a process commonly referred to as gastrulation. While substantial progress has been achieved in terms of characterizing the embryonic development of several model organisms, underlying principles of many early patterning processes nevertheless remain enigmatic. Despite the diversity of (pre-)gastrulating embryo and adult body shapes across the animal kingdom, the body axes, which are arguably the most fundamental features, generally remain identical between phyla. Recently there has been a renewed appreciation of ex vivo and in vitro embryo-like systems to model early embryonic patterning events. Here, we briefly review key examples and propose that similarities in morphogenesis and associated gene expression dynamics may reveal an evolutionarily conserved developmental mode as well as provide further insights into the role of external or extraembryonic cues in shaping the early embryo. In summary, we argue that embryo-like systems can be employed to inform previously uncharted aspects of animal body plan evolution as well as associated patterning rules. Metazoans display vast morphological diversity, yet body plans (box definitions) can universally be distilled to the presence of one to three body axes. Contrasted with protists, a characteristic feature is the existence of a species-specific embryonic phase in the respective life cycle. During its early stages, often collectively termed as gastrulation, simultaneous rearrangement and differentiation transforms a collection of embryonic (stem) cells into a complex, multilayered structure consisting of two (diploblasts) or three (triploblasts) germ layers organized along at least one primary body axis.Despite the conservation of gene regulatory networks that influence cell state and behavior, embryo geometry and overall tissue rearrangement dynamics can vary substantially during axial emergence . NeverthTraditionally, evolution of the primary axis has been studied from genomic and molecular perspectives in embryos across the animal kingdom, by examining the roles of, for instance, Wnt/β-catenin signaling and T-box genes that evolutionarily predate the cnidarian-bilaterian split . More reNematostella, Xenopus, zebrafish, mouse and human, it transpires that isolated ensembles from embryonic stem cells (ESCs) and ESC-like populations  universally harbor the capacity to self-organize (box definitions) into a rudimentary body plan with at least a primary axis. Notably, this may occur via developmental trajectories alternative to those in the respective native embryo. We hence argue that these observations could point toward a deeper, evolutionarily conserved developmental mode (box definitions) which cells exhibit as they are released from their species-specific geometrical arrangements and mechanochemical signaling environments  axis is ubiquitously identifiable at least in larval stages. This further arguably includes placozoans, demarcated along their respective upper and lower epithelia, the latter of which is used for feeding and exhibits expression of oral-associated genes . CnidariAs the second major group next to the diploblasts, all other extant animals are referred to as triploblasts or bilaterians. Despite there being considerable variability, the body plan of most bilaterian phyla features three distinct axes: Anteroposterior (AP), dorsoventral (DV), and mediolateral  as well as bilateral symmetry .Nematostella exhibits radial symmetry around the OA axis, bilateral symmetry takes over at late gastrula stages , studies have highlighted the convergent regulatory logic of AP and OA axis establishment via a Wnt/β-catenin-dependent system in deuterostomes and cnidarians . HoweverFor instance, in the context of bilaterian evolution, a direct correspondence between the cnidarian mouth and the bilaterian blastopore – a transient opening during the early gastrula stage of embryogenesis – is widely accepted. Yet, among bilaterians, there is substantial, species-specific secondary re-organization of mouth and anus formation .Altogether, in spite of the scarcity of experimental data in case of ctenophores, placozoans and sponges, it is hence still reasonable to assume that the AP axis of bilaterians, the OA axis of cnidarians, ctenophores and placozoans as well as the AP or AV axis of sponges constitute the most evolutionary ancient and thus main or primary body axes in these organisms.Sycon ciliatum sponge embryos, elevated Bra/T transcriptional activity is localized to a region undergoing a characteristic inversion event which shapes the thus emerging larva  and extraembryonic  – that generate the observed morphological diversity. We note that such ‘constraints’ do not imply less sophisticated developmental processes, on the contrary, in many species, external input is strictly required for complex morphogenesis to occur .Therefore, the study of ex vivo or in vitro systems can elucidate, on the one hand, shared principles and, on the other hand, provide an alternative angle: Removing stem cells (SCs) and SC-like populations from their native context potentially allows the disentanglement of such species-specific factors, thereby allowing cells to follow developmental trajectories guided by their inherent self-organizing capabilities .This concept is well exemplified by the various kinds of embryo-like structures, termed embryoids or embryonic organoids that have been developed recently from either embryonic or induced pluripotent stem cells (iPSCs) to recapitulate patterning events of initial embryogenesis until the onset of organogenesis .Box 1.Box definitions and abbreviations.Body plan A set of morphological features describing the body shape and structure of a given species. The most fundamental aspects of body plans, such as body axes, are conserved even across phyla.ESC-LPs Embryonic stem cell-like populations. We employ this term to summarize various kinds of cell populations which are capable of differentiating into the germ layers and assembling dynamically to generate a basic animal body plan. In this article, the term ESC-LP denotes pluripotent cells from different sources: induced (e.g. iPSCs) or present in vivo and spanning different states of the pluripotency spectrum (naïve versus primed ESCs). Such ESC-LPs are not specific to mammalian systems but can be present in species across the animal kingdom, including diploblasts and arthropods.Self-organization Emergence of order based on local interactions between different parts of a system that was initially disordered. Characteristic of such a process is feedback between components that amplifies the effects of local interactions or disturbance to the global level. In the context of aggregation experiments of ESCs and ESC-LPs discussed in this review, emergence of the primary axis can be considered self-organization. A related concept in biological systems is that of genetically encoded self-assembly environments and cues adopt a set of differentiation trajectories in terms of both morphogenesis and gene expression which are evolutionarily conserved across different species, in order to form patterned structures resembling actual embryos . A similar term, dynamical patterning or morphogenetic module (DPMs) refers to evolutionary conserved sets of gene products as well as their physical effects that alter the state, shape, size and arrangement of cells in a given population  development has had a long history of excising tissues from the native embryo and either transplanting them in a different embryo or different regions of the same embryo or culturing them as ex vivo explants over extended periods . While tXenopus, cell aggregates obtained by dissociating chordamesoderm (prospective notochord) from the early gastrula were shown to undergo cell sorting and convergent extension to establish an anteroposterior patterning in an elongated structure revealed through the expression of Bra/T (Xbra) and Chordin  in ng event , followeNematostella, fish, mouse and human embryos. While the precise mechanism remains to be elucidated, another commonality is the analogous dependence of primary axis formation on the initial number of cells in aggregates from two evolutionarily highly distant species: Larger re-aggregates from Nematostella gastrulae environments and associated cues.Nematostella, in which, during germ layer specification, trajectories of cells resemble those of cnidarians, such as hydrozoans. Yet, the end result is a functional animal, alike to one formed through normal Nematostella embryogenesis (It is noteworthy that this can indeed differ from the mode employed by the native embryo that uses a subset of (morphogenetic) trajectories available to the highly developmentally plastic embryonic cells . This plogenesis . In caseogenesis .Moreover, it was recently shown, through a transcriptional comparison of early mGastruloids to the mouse embryo, that while the emerging mesendodermal lineages in vitro are similar to their in vivo equivalents, AP axis and concomitant germ layer specification occurs in a parallel, but distinct manner, initiated by a more mesenchymal-like primed ESC population as opposed to the epithelial epiblast cells of the native embryo .Nematostella aboral halves, prior injection of Wnts is required to prompt the cells to develop into a functional animal (Complementary to the idea of ESC(-LP)s unraveling a conserved developmental mode under minimal culture conditions, is the observation that cells on a separate, advanced differentiation path can be coaxed to follow such basal trajectories when presented with the right factors. In re-aggregates from l animal . Conversl animal .Embryo-like structures also serve as a valuable tool to study the divergence of developmental trajectories in a systematic manner by exposing ESC(-LP)s to species-specific cues. These can be applied in manifold ways, for instance via adding early extraembryonic cells such as TSCs (trophoblast stem cells) or XEN (extraembryonic endoderm stem-) cells which in mammals give rise to tissues that closely interact with and instruct embryo patterning . FurtherIndeed, several recently described systems which implement such principles are allowing us to dive deeper into the question of how embryonic cells can generate a richer diversity of cell states and embryo-like morphology in the presence of aforementioned external cues.Single mESCs and TSCs, when grown together in a 3D Matrigel scaffold, mutually cooperate to assemble into structures, termed ETS embryos, highly reminiscent of the actual mouse embryo from E5.0 to 6.5 . They reWhereas in gastruloids the developing Bra/T pole or posterior seems to have no directional preference in the spherical aggregate, in ETS embryos, on the other hand, the presence of TSCs spatially confines the emergence of the this Bra/T domain at the boundary between embryonic and extraembryonic compartments, akin to the natural embryo. This role of the extraembryonic tissues is in congruence with the in vivo situation, where they provide not only the right signaling environment but also the crucial geometric cues that confer robustness to a developing embryo .This system was further complemented by adding a third cell type, XEN cells, which augment morphological similarity to in vivo mouse embryos by adding a visceral endoderm (VE)-like layer . MoreoveReplacing XEN cells with induced endodermal cells (transgenic mESCs featuring transient Gata4 upregulation) yields inducible ETX (iETX) embryos which more faithfully recapitulate particularly anterior morphogenetic events during mouse gastrulation . This apBlastoids, originally made from aggregated mESCs and TSCs, illustrate how the two cell types influence each other to form structures closely resembling mouse E3.5 blastocysts  with an outer trophoectoderm layer encapsulating the blastocoel, a fluid filled cavity, as well as the epiblast . It appeAlthough blastoids will not form bona fide embryos, they are capable of eliciting an implantation-like response in vivo, thereby constituting a potentially useful model system for studying mouse pre-gastrulation patterning dynamics. Other works also demonstrate the assembly of similar systems .In particular, EPS-blastoids are generated from extended pluripotent stem cells (EPSCs), pluripotent SCs cultured under conditions which enable both embryonic and extraembryonic developmental potential, as well as TSCs. These generate a primitive endoderm (PE)-like layer which, unlike normal blastoids, gives rise to cells expressing parietal endoderm markers. Moreover, under specific culture conditions, a subset of EPS-blastoids proceed to develop a post-implantation embryo-like morphology, similar to ETX embryos and E5.0-E5.5 cultured mouse embryos. The majority of these advanced EPS-blastoids also form an outer visceral endoderm (VE)-like layer.A series of very recent studies with hPSCs (induced or otherwise) demonstrated that also human blastocyst-like structures (hBlastoids) can be successfully obtained by either mixing hESCs with SCs from extra-embryonic lineages or via employing naïve hEPSCs, akin to the mouse model .By expanding on culture conditions of mGastruloids, further research efforts have coaxed these into recapitulating additional aspects of native embryo morphology. For example, a recent work reports the application of matrigel to generate somite-like structutures in conjunction with respective gene oscillation during mGastruloid elongation . A paralUpon administration of cardiogenic factors FGF, VEGF and ascorbic acid, mGastruloids reproducibly recapitulate cardiogenesis at their anterior end, giving rise a vascular-like network, first and second heart fields as well as ultimately a beating structure, resembling an actual embryonic heart . MoreoveAnother study details the generation of post-implantation epiblast-like (EPI) gastruloids which, in contrast to standard mGastruloids, specify anterior neural tissues upon additional Wnt inhibition via XAV939, while still developing an elongated morphology . This waEpi-aggregates were combined with TSC aggregates to generate structures labeled EpiTS embryoids that undergo axial elongation and concomitant patterning . AdditioIn a conceptually similar approach, two disparately sized ESC aggregates were fused after an initial aggregation and growth phase , with thRegarding human systems, it was recently demonstrated that pretreatment of human iPSCs with a Wnt agonist and FGF2 for 48 hr, followed by aggregation and maintenance in shaking culture, supplemented for 4 days with a growth factor cocktail  yields gastruloids, termed EMLO, that can be grown over several weeks . OverallBy employing a microfluidic device, researchers were able to recapitulate post-implantation embryo development prior to and during early gastrulation, encompassing epiblast luminogenesis, formation of the bipolar embryonic sac and PGC as well as primitive streak cell specification . To achiFollowing lumenogenesis, dorsal amniotic ectoderm-like cells were induced via localized application of BMP4. Ventral epiblast-like cells give rise to subpopulations expressing primitive streak markers such as BRA/T and PGC-associated genes. After 2–3 days in culture, cysts collapse due to emigration of ventral cells.Altogether, the above examples illustrate, in systems from both mouse and human, how embryo-like morphology can be recapitulated through mimicking of an extraembryonic environment or associated cues which provide morphogenetic instructions.In conjunction with the observed diversity in (pre-) gastrulation embryo shapes, comparison of in vitro and ex vivo systems to their in vivo counterparts may point toward an underlying developmental morphospace spanning a plethora of available trajectories .Since embryos of a given species generally develop under the same specific inputs, including geometry and extraembryonic environment, ESCs are developmentally biased and such alternative trajectories are revealed only when cells are removed from their native context and challenged by different external conditions . FurtherWhen comparing systems such as gastruloids with blastoids and ETS/ETX embryos, it becomes apparent that, while the latter (mostly) faithfully recapitulate embryo geometry and morphogenesis, they are, at the present time, not able to develop beyond the in vivo equivalent of embryonic stage E7.0-E7.5. On the other hand, gastruloids do not look like actual embryos and undergo a more abstracted morphogenesis, yet they are capable of reaching the partial  equivalent of E9.0 (in case of the mESC-based system).These observations raise an interesting point: It seems that by employing additional extraembryonic cell types or, in general terms, by mimicking a set of in vivo cues, resulting embryo-shaped artificial systems remain more constrained in their developmental potential. This is likely because, once having assumed an embryo-like developmental mode early during their respective morphogenesis, such structures would require further precise, spatially and temporally allocated inputs, just like the in vivo counterpart, in order to facilitate successive patterning events. In this context, it has to be mentioned that with advances in biomaterials and biotechnology, it should be possible to mimic extraembryonic environments more closely in the future .Gastruloid-like aggregates from pure ESCs or ESC-LPs, on the other hand, might represent a more unconstrained system, which is what enables them to develop beyond the equivalent of initial gastrulation, and therefore unveil the extent of inherent self-organizational capabilities of the source cells.In summary, the wide range of trajectories displayed by ESCs and ESC-LPs in vitro converge onto an aforementioned conserved developmental mode under minimal conditions and collapse partly onto their native trajectories  when provided with external inputs to recapitulate species-specific external environments , facilitating increased morphological resemblance to the native embryo.Characterizing this fundamental mode may perhaps ultimately provide insights into the origin and evolution of animal body plans as well as how the complexity of animal shapes during peri-gastrulation development may have arisen via distinct extraembryonic factors. In order to properly address this from a comprehensive evolutionarily perspective, however, more phylum coverage is required, dictating the need for novel minimal in vitro embryo-like systems from, for instance, diploblasts and protostomes.Concerning the significance of extraembryonic environments, it could be argued that, from the multitude of ways that pre-gastrulating embryos have at their disposal to establish their initial body plan, refinement of a species-specific developmental mode as well as corresponding (extra-)embryonic morphology over the course of evolution ensures robustness  : Those dWhile this hypothesis remains unverified in metazoan embryos at large, a recent study finds that diversification of insect egg size and shape, traits which vary greatly across species, are driven by shifts in the respective oviposition microenvironments, that is, where the eggs are laid . LikewisNematostella or Starfish larva as opposed to a fish, mouse, or human from isolated ESC-like cells.Such external or extraembryonic environments and their complexity vary greatly across species and constitute a substantial challenge to replicate in vitro. It is natural to speculate that this could be one of the reasons why it is more straightforward to generate a functional Nonetheless, comparing these different kinds of embryo-like structures should therefore provide tangible clues as to which aspects of embryonic development or, more precisely, axis formation and associated morphogenesis are inherent to ESCs and which parts strictly require extraembryonic input.hourglass model delineates a phylotypic period at mid-embryogenesis during which common anatomical features (the basic body plan) for each respective phylum are established , referring to a transition period between early and late stages of conserved gene expression which is characterized by phylum-specific activity of gene networks . This phThe conserved developmental mode that we propose here, on the other hand, applies to ESCs across phyla . Moreover, this mode entails the establishment of primary axial identity from an initially homogeneous or disorganized cell population, whereas the MDT and phylotypic period span the organogenesis phase of embryonic development, at which point the respective body axes are already specified.However, it can be conjectured that future study of embryo-like systems may reveal further developmental modes, associated with the emergence of corresponding dynamical patterning modules. It is lTaken together, establishment of new in vitro embryo-like systems in conjunction with refinement of existing ones and investigation of their displayed initial patterning dynamics will help to assess if or to which degree aforementioned conserved developmental modes exist. Hence, integration and comparison of such studies with data from actual embryos should aid to more concisely delineate underlying mechanisms of primary axis and subsequent initial body plan specification in cell ensembles as well as to elucidate early (pre- and peri-gastrulation) embryo morphology diversification during evolution.