Clonal Origin and Lineage Ambiguity in Mixed Neuroendocrine Carcinoma of the Uterine Cervix.

Small-cell neuroendocrine carcinoma (SCNEC) of the cervix is a rare disease characterized by a high incidence of mixed tumors with other types of cancer. The mechanism underlying this mixed phenotype is not well understood. This study established a panel of organoid lines from patients with SCNEC of the cervix and ultimately focused on one line, which retained a mixed tumor phenotype, both in vitro and in vivo. Histologically, both organoids and xenograft tumors showed distinct differentiation into either SCNEC or adenocarcinoma in some regions and ambiguous differentiation in others. Tracking single cells indicated the existence of cells with bipotential differentiation toward SCNEC and adenocarcinomas. Single-cell transcriptional analysis identified three distinct clusters: SCNEC-like, adenocarcinoma-like, and a cluster lacking specific differentiation markers. The expression of neuroendocrine markers was enriched in the SCNEC-like cluster but not exclusively. Human papillomavirus 18 E6 was enriched in the SCNEC-like cluster, which showed higher proliferation and lower levels of the p53 pathway. After treatment with anticancer drugs, the expression of adenocarcinoma markers increased, whereas that of SCNEC decreased. Using a reporter system for keratin 19 expression, changes in the differentiation of each cell were shown to be associated with the shift in differentiation induced by drug treatment. These data suggest that mixed SCNEC/cervical tumors have a clonal origin and are characterized by an ambiguous and flexible differentiation state.

Inhibition of the bone morphogenetic protein pathway suppresses tumor growth through downregulation of epidermal growth factor receptor in MEK/ERK-dependent colorectal cancer.

The bone morphogenetic protein (BMP) pathway promotes differentiation and induces apoptosis in normal colorectal epithelial cells. However, its role in colorectal cancer (CRC) is controversial, where it can act as context-dependent tumor promoter or tumor suppressor. Here we have found that CRC cells reside in a BMP-rich environment based on curation of two publicly available RNA-sequencing databases. Suppression of BMP using a specific BMP inhibitor, LDN193189, suppresses the growth of select CRC organoids. Colorectal cancer organoids treated with LDN193189 showed a decrease in epidermal growth factor receptor, which was mediated by protein degradation induced by leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) expression. Among 18 molecularly characterized CRC organoids, suppression of growth by BMP inhibition correlated with induction of LRIG1 gene expression. Notably, knockdown of LRIG1 in organoids diminished the growth-suppressive effect of LDN193189. Furthermore, in CRC organoids, which are susceptible to growth suppression by LDN193189, simultaneous treatment with LDN193189 and trametinib, an FDA-approved MEK inhibitor, resulted in cooperative growth inhibition both in vitro and in vivo. Taken together, the simultaneous inhibition of BMP and MEK could be a novel treatment option in CRC cases, and evaluating in vitro growth suppression and LRIG1 induction by BMP inhibition using patient-derived organoids could offer functional biomarkers for predicting potential responders to this regimen.

Abnormality of apico-basal polarity in adenocarcinoma.

Apico-basal polarity is a fundamental property of the epithelium that functions as a barrier, holds cells together, and determines the directions of absorption and secretion. Apico-basal polarity is regulated by extracellular matrix-integrin binding and downstream signaling pathways, including focal adhesion kinase, rouse-sarcoma oncogene (SRC), and RHO/RHO-associated kinase (ROCK). Loss of epithelial cell polarity plays a critical role in the progression of cancer cells. However, in differentiated carcinomas, polarity is not completely lost but dysregulated. Recent progress with a three-dimensional culture of primary cancer cells allowed for studies of the mechanism underlying the abnormality of polarity in differentiated cancers, including flexible switching of polarity status in response to the microenvironment. Invasive micropapillary carcinoma (MPC) is one of the histopathological phenotypes of adenocarcinoma, which is characterized by inverted polarity. Aberrant activation of RHO-ROCK signaling plays a critical role in the MPC phenotype. Establishing in vitro models will contribute to future drug targeting of the abnormal polarity status in cancer.

Polarity switching of ovarian cancer cell clusters via SRC family kinase is involved in the peritoneal dissemination.

Peritoneal dissemination is a predominant pattern of metastasis in patients with advanced ovarian cancer. Despite recent progress in the management strategy, peritoneal dissemination remains a determinant of poor ovarian cancer prognosis. Using various histological types of patient-derived ovarian cancer organoids, the roles of the apicobasal polarity of ovarian cancer cell clusters in peritoneal dissemination were studied. First, it was found that both ovarian cancer tissues and ovarian organoids showed apicobasal polarity, where zonula occludens-1 (ZO-1) and integrin beta 4 (ITGB4) served as markers for apical and basal sides, respectively. The organoids in suspension culture, as a model of cancer cell cluster floating in ascites, showed apical-out/basal-in polarity status, while once embedded in extracellular matrix (ECM), the organoids switched their polarity to apical-in/basal-out. This polarity switch was accompanied by the SRC kinase family (SFK) phosphorylation and was inhibited by SFK inhibitors. SFK inhibitors abrogated the adherence of the organoids onto the ECM-coated plastic surface. When the organoids were seeded on a mesothelial cell layer, they cleared and invaded mesothelial cells. In vivo, dasatinib, an SFK inhibitor, suppressed peritoneal dissemination of ovarian cancer organoids in immunodeficient mice. These results suggest SFK-mediated polarity switching is involved in peritoneal metastasis. Polarity switching would be a potential therapeutic target for suppressing peritoneal dissemination in ovarian cancer.

Aberrant activation of Rho/ROCK signaling in impaired polarity switching of colorectal micropapillary carcinoma.

Micropapillary carcinoma (MPC) is a morphologically distinctive form of carcinoma, composed of small nests of cancer cells surrounded by lacunar spaces. Invasive MPC is associated with poor prognosis. The nests of tumor cells in MPC reportedly exhibit reverse polarity, although the molecular mechanisms underlying MPC patterns are poorly understood. Using the cancer tissue-originated spheroid (CTOS) method, we previously reported polarity switching in colorectal cancer (CRC). When cultured in suspension, the apical membrane promptly switches from the outside surface of the CTOSs to the surface of the lumen inside the CTOSs under extracellular matrix (ECM)-embedded conditions, and vice versa. Here, we investigated two CTOS lines from CRC patient tumors with MPC lesions. Xenograft tumors from the CTOSs exhibited the MPC phenotype. The MPC-CTOSs did not switch polarity in vitro. Time-course analysis of polarity switching using real-time imaging of the apical membrane revealed that local switching was continually propagated in non-MPC-CTOSs, while MPC-CTOSs were unable to complete the process. Integrin ß4 translocated to the outer membrane when embedded in ECM in both MPC and non-MPC-CTOSs. Protein levels, as well as the active form of RhoA, were higher in MPC-CTOSs. The suppression of RhoA activity by GAP overexpression enabled MPC-CTOSs to complete polarity switching both in vitro and in vivo, while overexpression of active RhoA did not affect polarity switching in non-MPC-CTOSs. Pretreatment with a ROCK inhibitor enabled MPC-CTOSs to complete polarity switching both in vitro and in vivo, although delayed treatment after becoming embedded in ECM failed to do so. Thus, the inability to switch polarity might be a cause of MPC, in which the aberrant activation of RhoA plays a critical role. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Loss of the cystine/glutamate antiporter in melanoma abrogates tumor metastasis and markedly increases survival rates of mice.

The cystine/glutamate antiporter, system xc- , is essential for the efficient uptake of cystine into cells. Interest in the mechanisms of system xc- function soared with the recognition that system xc- presents the most upstream node of ferroptosis, a recently described form of regulated necrosis relevant for degenerative diseases and cancer. Since targeting system xc- hold the great potential to efficiently combat tumor growth and metastasis of certain tumors, we disrupted the substrate-specific subunit of system xc- , xCT (SLC7A11) in the highly metastatic mouse B16F10 melanoma cell line and assessed the impact on tumor growth and metastasis. Subcutaneous injection of tumor cells into the syngeneic B16F10 mouse melanoma model uncovered a marked decrease in the tumor-forming ability and growth of KO cells compared to control cell lines. Strikingly, the metastatic potential of KO cells was markedly reduced as shown in several in vivo models of experimental and spontaneous metastasis. Accordingly, survival rates of KO tumor-bearing mice were significantly prolonged in contrast to those transplanted with control cells. Analyzing the in vitro ability of KO and control B16F10 cells in terms of endothelial cell adhesion and spheroid formation revealed that xCT expression indeed plays an important role during metastasis. Hence, system xc- emerges to be essential for tumor metastasis in mice, thus qualifying as a highly attractive anticancer drug target, particularly in light of its dispensable role for normal life in mice.

Small subset of Wnt-activated cells is an initiator of regrowth in colorectal cancer organoids after irradiation.

Most colorectal cancers (CRCs) are differentiated adenocarcinomas, which maintain expression of both stemness and differentiation markers. This observation suggests that CRC cells could retain a regeneration system of normal cells upon injury. However, the role of stemness in cancer cell regeneration after irradiation is poorly understood. Here, we examined the effect of radiation on growth, stemness, and differentiation in organoids derived from differentiated adenocarcinomas. Following a sublethal dose of irradiation, proliferation and stemness markers, including Wnt target genes, were drastically reduced, but differentiation markers remained. After a static growth phase after high dose of radiation, regrowth foci appeared; these consisted of highly proliferating cells that expressed stem cell markers. Radiosensitivity and the ability to form foci differed among the cancer tissue-originated spheroid (CTOS) lines examined and showed good correlation with in vivo radiation sensitivity. Pre-treating organoids with histone deacetylase inhibitors increased radiation sensitivity; this increase was accompanied by the suppression of Wnt signal-related gene expression. Accordingly, Wnt inhibitors increased organoid radiosensitivity. These results suggested that only a small subset of, but not all, cancer cells with high Wnt activity at the time of irradiation could give rise to foci formation. In conclusion, we established a radiation sensitivity assay using CRC organoids that could provide a novel platform for evaluating the effects of radiosensitizers on differentiated adenocarcinomas in CRC.

Dedifferentiation of neuroendocrine carcinoma of the uterine cervix in hypoxia.

Neuroendocrine carcinoma of small cell type (SCNEC) is a rare pathological subtype in cervical cancer, which has a worse prognosis than other histological cell types. Due to its low incidence and the lack of experimental platforms, the molecular characteristics of SCNEC in the cervix remain largely unknown. Using the cancer tissue-originated spheroid (CTOS) method-an ex vivo 3D culture system that preserves the differentiation status of the original tumors-we established a panel of CTOS lines of SCNEC. We demonstrated that xenograft tumors and CTOSs, respectively, exhibited substantial intra-tumor and intra-CTOS variation in the expression levels of chromogranin A (CHGA), a neuroendocrine tumor marker. Since hypoxia affects differentiation in various tumors and in stem cells, we also investigated how hypoxia affected neuroendocrine differentiation of SCNEC of the uterine cervix. In the CTOS line cerv21, hypoxia suppressed expression of the neuroendocrine markers CHGA and synaptophysin (SYP). Flow cytometry analysis using CD99 (a membrane protein marker of SCNEC) revealed decreased CD99 expression in a subset of cells under hypoxic conditions. These expression changes were attenuated by HIF-1α knockdown, and by a Notch inhibitor, suggesting that these molecules played a role in the regulation of neuroendocrine differentiation. The examined SCNEC markers were suppressed under hypoxia in multiple CTOS lines. Overall, our present results indicated that neuroendocrine differentiation in SCNEC of the uterus is a variable phenotype, and that hypoxia may be one of the factors regulating the differentiation status.

Shortcuts to intestinal carcinogenesis by genetic engineering in organoids.

Inactivation of the Adenomatous polyposis coli (APC) gene is an initiating and the most relevant event in most sporadic cases of colorectal cancer, providing a rationale for using Apc-mutant mice as the disease model. Whereas carcinogenesis has been observed only at the organism level, the recent development of the organoid culture technique has enabled long-term propagation of intestinal stem cells in a physiological setting, raising the possibility that organoids could serve as an alternative platform for modeling colon carcinogenesis. Indeed, it is demonstrated in the present study that lentivirus-based RNAi-mediated knockdown of Apc in intestinal organoids gave rise to subcutaneous tumors upon inoculation in immunodeficient mice. Reconstitution of common genetic aberrations in organoids resulted in development of various lesions, ranging from aberrant crypt foci to full-blown cancer, recapitulating multi-step colorectal tumorigenesis. Due to its simplicity and utility, similar organoid-based approaches have been applied to both murine and human cells in many investigations, to gain mechanistic insight into tumorigenesis, to validate putative tumor suppressor genes or oncogenes, and to establish preclinical models for drug discovery. In this review article, we provide a multifaceted overview of these types of approaches that will likely accelerate and advance research on colon cancer.

Synthesis and biological evaluation of glucose conjugated phthalocyanine as a second-generation photosensitizer.

Photodynamic therapy (PDT) is a treatment method using light and photosensitizers (PSs), which is categorized as a non-invasive surgery treatment for cancers. When the tumor is exposed to a specific light, the PSs become active and generate reactive oxygen species (ROS), mainly singlet oxygen which kills nearby cancer cells. PDT is becoming more widely recognized as a valuable treatment option for localized cancers and pre-cancers of skin as it has no long-term effects on the patient. But, due to the limited penetration rate of light into the skin and other organs, PDT can't be used to treat large cancer cells or cancer cells that have grown deeply into the skin or other organs. Hence, in this study, our focus centers on synthesizing glucose-conjugated phthalocyanine (Pc) compatible with near-infrared (NIR) irradiation as second-generation photosensitizer, so that PDT can be used in a wider range to treat cancers without obstacles.

Fascin protein stabilization by miR-146a implicated in the process of a chronic inflammation-related colon carcinogenesis model.

OBJECTIVE: In sporadic colon tumors, multistep process of well-known genetic alterations accelerates carcinogenesis; however, this does not appear to be the case in inflammation-related ones. We previously established a model of inflammation-related colon carcinogenesis using human colonic adenoma cells, and identified fascin as a driver gene of this process. We analyzed the microRNAs involved in the stable fascin expression in colon adenocarcinoma cells. MATERIALS AND METHODS: miRNA microarray analysis was performed using FPCK-1-1 adenoma cells and its-derived FPCKpP1-4 adenocarcinoma cells through chronic inflammation. To assess the involvement of miRNA in the inflammation-related carcinogenesis, sphere-forming ability, expression of colon cancer stemness markers, and stability of fascin protein via the proteasome using tough decoy RNA technique. RESULTS: We found that 17 miRNAs including miR-146a were upregulated and 16 miRNAs were downregulated in FPCKpP1-4 adenocarcinoma cells. We revealed that miR-146a in the adenocarcinoma cells brought about acquisition of sphere formation, cancer stemness, and inhibition of proteasomal degradation of the fascin protein. CONCLUSIONS: We found that stable fascin expression is brought about via the inhibition of proteasome degradation by miR-146a in the process of a chronic inflammation-related colon carcinogenesis.

Design, synthesis, and biological evaluation of a highly water-soluble psoralen-based photosensitizer.

In recent years, photodynamic therapy (PDT) has been approved for treating various medical conditions, including cancer. PDT is a treatment that employs a particular drugs, called photosensitizers which work along with specific light source. The growth of this medical industry is expanding as it is another promising alternative to treat cancer which lessen the burden of treatments in patients. This includes the benefits of minimally invasive procedures and delivering high accuracy in targeting mutations. In recent two decades, cancer researchers have produced remarkable studies on developing photosensitizers that enhance understanding of biology and genetics of cancer. It is unfortunate that not all PDT can work as well as other profound treatment because PDT has various limitations like PDT leads photosensitivity reaction that arises when the photosensitizer remains in the body for a long period of time. In this paper, our studies centers on synthesizing a highly soluble photosensitizing agent with improved effectiveness on detecting cancer cells.

Genetic reconstitution of tumorigenesis in primary intestinal cells.

Animal models for human colorectal cancer recapitulate multistep carcinogenesis that is typically initiated by activation of the Wnt pathway. Although potential roles of both genetic and environmental modifiers have been extensively investigated in vivo, it remains elusive whether epithelial cells definitely require interaction with stromal cells or microflora for tumor development. Here we show that tumor development could be simply induced independently of intestinal microenvironment, even with WT murine primary intestinal cells alone. We developed an efficient method for lentiviral transduction of intestinal organoids in 3D culture. Despite seemingly antiproliferative effects by knockdown of adenomatous polyposis coli (APC), we managed to reproducibly induce APC-inactivated intestinal organoids. As predicted, these organoids were constitutively active in the Wnt signaling pathway and proved tumorigenic when injected into nude mice, yielding highly proliferative tubular epithelial glands accompanied by prominent stromal tissue. Consistent with cellular transformation, tumor-derived epithelial cells acquired sphere formation potential, gave rise to secondary tumors on retransplantation, and highly expressed cancer stem cell markers. Inactivation of p53 or phosphatase and tensin homolog deleted from chromosome 10, or activation of Kras, promoted tumor development only in the context of APC suppression, consistent with earlier genetic studies. These findings clearly indicated that genetic cooperation for intestinal tumorigenesis could be essentially recapitulated in intestinal organoids without generating gene-modified mice. Taken together, this in vitro model for colon cancer described herein could potentially provide unique opportunities for carcinogenesis studies by serving as a substitute or complement to the currently standard approaches.

Fascin regulates chronic inflammation-related human colon carcinogenesis by inhibiting cell anoikis.

By a proteomics-based approach, we identified an overexpression of fascin in colon adenocarcinoma cells (FPCKpP-3) that developed from nontumorigenic human colonic adenoma cells (FPCK-1-1) and were converted to tumorigenic by foreign-body-induced chronic inflammation in nude mice. Fascin overexpression was also observed in the tumors arising from rat intestinal epithelial cells (IEC 6) converted to tumorigenic in chronic inflammation which was induced in the same manner. Upregulation of fascin expression in FPCK-1-1 cells by transfection with sense fascin cDNA converted the cells tumorigenic, whereas antisense fascin-cDNA-transfected FPCKpP-3 cells reduced fascin expression and lost their tumor-forming ability in vivo. The tumorigenic potential by fascin expression was consistent with their ability to survive and grow in the three-dimensional multicellular spheroids. We found that resistance to anoikis (apoptotic cell death as a consequence of insufficient cell-to-substrate interactions), which is represented by the three-dimensional growth of solid tumors in vivo, was regulated by fascin expression through caspase-dependent apoptotic signals. From these, we demonstrate that fascin is a potent suppressor to caspase-associated anoikis and accelerator of the conversion of colonic adenoma cells into adenocarcinoma cells by chronic inflammation.

Tracking the growth fate of single cells and isolating slow-growing cells in human colorectal cancer organoids.

Patient-derived tumor organoids are three-dimensionally cultured cancer cells that enable a suitable platform for studying heterogeneity and plasticity of cancer. We present a protocol for tracking the growth fate of single cells and isolating slow-growing cells in human colorectal cancer organoids. We describe steps for organoid preparation and culturing using the cancer-tissue-originating spheroid method, maintaining cell-cell contact throughout. We then detail a single-cell-derived spheroid-forming and growth assay, confirming single-cell plating, monitoring growth over time, and isolating slow-growing cells. For complete details on the use and execution of this protocol, please refer to Coppo et al.1.

Oxygen metabolism analysis of a single organoid for non-invasive discrimination of cancer subpopulations with different growth capabilities.

Heterogeneous nature is a pivotal aspect of cancer, rendering treatment problematic and frequently resulting in recurrence. Therefore, advanced techniques for identifying subpopulations of a tumour in an intact state are essential to develop novel screening platforms that can reveal differences in treatment response among subpopulations. Herein, we conducted a non-invasive analysis of oxygen metabolism on multiple subpopulations of patient-derived organoids, examining its potential utility for non-destructive identification of subpopulations. We utilised scanning electrochemical microscopy (SECM) for non-invasive analysis of oxygen metabolism. As models of tumours with heterogeneous subpopulations, we used patient-derived cancer organoids with a distinct growth potential established using the cancer tissue-originated spheroid methodology. Scanning electrochemical microscopy measurements enabled the analysis of the oxygen consumption rate (OCR) for individual organoids as small as 100 µm in diameter and could detect the heterogeneity amongst studied subpopulations, which was not observed in conventional colorectal cancer cell lines. Furthermore, our oxygen metabolism analysis of pre-isolated subpopulations with a slow growth potential revealed that oxygen consumption rate may reflect differences in the growth rate of organoids. Although the proposed technique currently lacks single-cell level sensitivity, the variability of oxygen metabolism across tumour subpopulations is expected to serve as an important indicator for the discrimination of tumour subpopulations and construction of novel drug screening platforms in the future.

De-differentiation in cultures of organoids from luminal-type breast cancer is restored by inhibition of NOTCH signaling.

Estrogen receptor (ER) expression in breast cancer can change during progression and the treatment, but the mechanism has not been well studied. In this study, we successfully prepared organoids from samples obtained from 33 luminal-type breast cancer patients and studied their ER expression. The expression status was well maintained in primary organoids, whereas it decreased after passaging in most of the cases. In fact, the studied organoid lines were classified into those that retained a high level of ER expression (9%), those that completely lost it (9%), and those that repressed it to varying degrees (82%). In some cases, the ER expression was suddenly and drastically decreased after passaging. Marker protein immunohistochemistry revealed that after passaging, the differentiation status shifted from a luminal- to a basal-like status. Differentially expressed genes suggested the activation of NOTCH signaling in the passaged organoids, wherein a NOTCH inhibitor was able to substantially rescue the decreased ER expression and alter the differentiation status. Our findings suggest that the differentiation status of luminal-type cancer cells is quite flexible, and that by inhibiting the NOTCH signaling we can preserve the differentiation status of luminal-type breast cancer organoids.

Electrochemical sensing of oxygen metabolism for a three-dimensional cultured model with biomimetic vascular flow.

Microphysiological systems (MPSs) with three-dimensional (3D) cultured models have attracted considerable interest because of their potential to mimic human health and disease conditions. Recent MPSs have shown significant advancements in engineering perfusable vascular networks integrated with 3D culture models, realizing a more physiological environment in vitro; however, a sensing system that can monitor their activity under biomimetic vascular flow is lacking. We designed an open-top microfluidic device with sensor capabilities and demonstrated its application in analyzing oxygen metabolism in vascularized 3D tissue models. We first validated the platform by using human lung fibroblast (hLF) spheroids. Then, we applied the platform to a patient-derived cancer organoid and evaluated the changes in oxygen metabolism during drug administration through the vascular network. We found that the platform could integrate a perfusable vascular network with 3D cultured cells, and the electrochemical sensor could detect the change in oxygen metabolism in a quantitative, non-invasive, and real-time manner. This platform would become a monitoring system for 3D cultured cells integrated with a perfusable vascular network.

Ultra-Rapid and Specific Gelation of Collagen Molecules for Transparent and Tough Gels by Transition Metal Complexation.

Collagen is the most abundant protein in the human body and one of the main components of stromal tissues in tumors which have a high elastic modulus of over 50 kPa. Although collagen has been widely used as a cell culture scaffold for cancer cells, there have been limitations when attempting to fabricate a tough collagen gel with cells like a cancer stroma. Here, rapid gelation of a collagen solution within a few minutes by transition metal complexation is demonstrated. Type I collagen solution at neutral pH shows rapid gelation with a transparency of 81% and a high modulus of 1,781 kPa by mixing with K2 PtCl4 solution within 3 min. Other transition metal ions also show the same rapid gelation, but not basic metal ions. Interestingly, although type I to IV collagen molecules show rapid gelation, other extracellular matrices  do not exhibit this phenomenon. Live imaging of colon cancer organoids in 3D culture indicates a collective migration property with modulating high elastic modulus, suggesting activation for metastasis progress. This technology will be useful as a new class of 3D culture for cells and organoids due to its facility for deep-live observation and mechanical stiffness adjustment.

Distinct but interchangeable subpopulations of colorectal cancer cells with different growth fates and drug sensitivity.

Dynamic changes in cell properties lead to intratumor heterogeneity; however, the mechanisms of nongenetic cellular plasticity remain elusive. When the fate of each cell from colorectal cancer organoids was tracked through a clonogenic growth assay, the cells showed a wide range of growth ability even within the clonal organoids, consisting of distinct subpopulations; the cells generating large spheroids and the cells generating small spheroids. The cells from the small spheroids generated only small spheroids (S-pattern), while the cells from the large spheroids generated both small and large spheroids (D-pattern), both of which were tumorigenic. Transition from the S-pattern to the D-pattern occurred by various extrinsic triggers, in which Notch signaling and Musashi-1 played a key role. The S-pattern spheroids were resistant to chemotherapy and transited to the D-pattern upon drug treatment through Notch signaling. As the transition is linked to the drug resistance, it can be a therapeutic target.