The differentiation status of luminal-type breast cancer–derived organoids can be altered in vitro through the activation of NOTCH signaling

doi:10.21203/rs.3.rs-2232037/v1

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The differentiation status of luminal-type breast cancer–derived organoids can be altered in vitro through the activation of NOTCH signaling

https://doi.org/10.21203/rs.3.rs-2232037/v1

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Background

Drug therapy of luminal-type breast cancer has advanced remarkably; however, its effectiveness continues to be limited by primary and acquired treatment resistance. To identify the means of overcoming such resistance, in vitro disease models that faithfully reproduce the characteristics of original patient tumors must be developed. Although the three-dimensional culturing of breast cancer cells has been improved, the changes in differentiation status and underlying mechanisms require further elucidation.

Methods

The organoids from 33 luminal-type breast cancers were prepared using a method developed by us. Using immunohistochemistry and gene expression profiling, the levels and time courses of estrogen receptor, progesterone receptor, Ki67, and differentiation marker expression were monitored during culture as well as the variation among the patients and individual organoids. Furthermore, we examined the effect of NOTCH inhibitor treatment on differentiation status.

Results

We successfully prepared organoids from samples obtained from 33 patients with luminal-type breast cancer 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.

Conclusions

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.

Breast cancer

Estrogen receptor

Organoids

Notch

Breast cancer is the most frequently diagnosed type of cancer and the leading cause of cancer-associated death in women worldwide [1]. The estrogen receptor (ER) is the major driver in breast cancer and is expressed in 75% of the overall cases [2]. ER-positive luminal-type breast cancer is treated with hormone therapy [3], but its effectiveness is limited by the high rates of primary and acquired treatment resistance [4]. Recently, small-molecule inhibitors of the CDK4/6 cell-cycle kinases have exhibited clinical efficacy in ER-positive metastatic breast cancer, although their effects are also limited by primary and acquired treatment resistance [5]. To identify ways to overcome the resistance to these therapies, it is particularly important to develop in vitro disease models in which critical characteristics of the original patient tumors (such as heterogeneity and plasticity) are faithfully reproduced.

Cell lines established in two-dimensional culture conditions with the use of serum-containing medium have the advantage of being easy to hand; however, relatively few ER-positive breast cancer cell lines have been established so far, and the MCF7 cells, established from a patient’s pleural effusion in 1973, have been heavily used as a model of ER-positive breast cancer [6]. Moreover, there is a concern that those established cell lines have drifted away from the phenotype of the originating tumor [7]. The three-dimensional (3D) culturing of breast cancer cells has been recently improved owing to the use of laminin-rich extracellular matrix [8] and the addition of ‘niche factors’ in the culture medium [9]; a process leading to the generation of “organoids.” Despite the improvement of the success rate as a result of the adoption of the organoid-generating methods, it has been suggested that the ER expression diminishes in more than 60% of the organoids prepared from luminal-type breast cancers [9–12]. The process and the mechanisms underlying this decrease of the ER expression in the organoid culture have not been well studied yet.

The study of organoids is now widely conducted and applied in cancer research [13]; however, little attention has been paid to the methodology employed for preparing tumor-derived organoids. The typical preparation process can be summarized using the phrase “mechanically and chemically digested,” i.e., indicating that the cells are digested into single or nearly single cells. In contrast, we developed the CTOS (cancer tissue-originated spheroid) method [14], in which the cancer cells are retrieved from patient tumors in the form of clusters.

In this study, we prepared organoids from breast cancers according to the CTOS method, we cultured them by using the reported medium and the cell matrix [9], and we investigated the details of the decrease of the ER expression during the culturing process (including the extent, time course, and variation among the patients) as well as the underlying molecular mechanisms.

Patient samples

This study was approved by the institutional ethics committees of Kyoto University (R2220, R2444) and Kansai Medical University (2019245, 2021090). With written informed consent, tumor specimens were obtained from patients who underwent needle biopsy or surgery at the Kansai Medical University. A total of 39 breast cancer samples were obtained from 37 patients (Table S1). Of these, 18 were biopsy specimens and 21 corresponded to surgically removed specimens. Two samples were from the same patients, and were obtained from preoperative biopsy and from surgery. Four normal breast tissues were obtained from surgically removed breasts (from parts that were located in good distance from the tumors).

Organoid preparation

The preparation of the organoids from patient samples was performed through the employment of a modified CTOS method [14]. For the mechanical dissociation of the tissues, we used a sharp blade (61-S, FEATHER, Osaka, Japan) to minimize the tissue crushing. The tissues were then digested with a 0.26 U/mL Liberase DH solution (Roche Diagnostics, Mannheim, Germany) at 37℃, for 1 h, and the cancer cell clusters were manually picked up under the microscope, sometimes by using 27G needles to retrieve the cancer cell clusters trapped in undigested tissue fragments. Finally, the obtained clusters were embedded in a Matrigel® growth factor reduced (GFR) basement membrane matrix (Corning®, Corning, NY, USA), and were cultured in the medium as previously described [9].

Organoid culturing

Breast cancer and mammary organoids were embedded in 100% Matrigel GFR droplets on untreated tissue culture dishes (AGC techno glass, Shizuoka, Japan), and were overlaid with culture medium consisting of advanced DMEM/F12 (Thermo Fisher Scientific, Waltham, MA, USA) that was supplemented with 250 ng/mL R-spondin-1 (R&D systems Wiesbaden-Nordenstadt, Germany), 5 nM Heregulin β-1 (Peprotech, Rocky Hill, NJ, USA), 5 ng/mL FGF-7 (Peprotech), 20 ng/mL FGF-10 (Peprotech), 5 ng/mL EGF (R&D systems), 100 ng/mL Noggin (Peprotech), 500 nM A83-01 (Tocris Bioscience, Bristol, UK), 5 µM Y-27632 (Selleck Chemicals, Houston, TX, USA), 500 nM SB202190 (Selleck Chemicals), 1× B27 (Thermo Fisher Scientific), 1.25 mM N-acetylcysteine (Merck Millipore, Darmstadt, Germany), 5 mM nicotinamide (Sigma-Aldrich, St Louis, MO, USA), 1× GlutaMAX (Thermo Fisher Scientific), 10 mM HEPES (Fujifilm Wako Pure Chemical Corporation, Osaka, Japan), and 100 U/mL penicillin/streptomycin (Thermo Fisher Scientific). For NOTCH inhibition, 25 µM DAPT (Sigma-Aldrich) was added to the culture medium or buffers. The organoids were incubated in a 5% CO₂ incubator at 37℃, and their medium was exchanged every 3−4 days. For their expansion, breast cancer organoids were passaged through dissociation into small cell clusters (but not into single cells) after incubating with TrypLE™ Express Enzyme (Thermo Fisher Scientific) at 37°C for 10 min and pipetting with a P200 PIPETMAN (Middleton, WI, USA) for 20 times. The small cell clusters were re-embedded in 5 µL droplets of 100% Matrigel GFR and were overlaid with culture medium.

Immunohistochemistry

The immunohistochemistry of formalin-fixed, paraffin-embedded clinical samples and organoids was performed as previously described [15]. The primary antibodies for following proteins were used: Estrogen receptor (ER; Roche Diagnostics), progesterone receptor (PGR; Roche Diagnostics), cytokeratin 5 (CK5; Abcam, Cambridge, MA, USA), cytokeratin 8 (CK8; DSHB, Iowa city, IA, USA), cytokeratin 19 (CK19; Abcepta, San Diego, CA, USA), and Ki67 (Cell Signaling Technology, Beverly, MA, USA). The nuclei were stained by 4',6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific). Images were acquired by BX50 through the use of the CellSens standard imaging software (Olympus, Tokyo, Japan). In some cases (KAN1, KAN6, KAN7, KAN8, and KAN21), a wholemount staining was performed due to the fact that only a small number of the organoids was obtained. For the wholemount staining, the organoids were retrieved from the Matrigel by digesting the gel with a Cell Recovery Solution (Corning) at 4℃ for 60 min, were fixed with acetone/methanol (1:1) at 4°C for 45 min, and were permeabilized with phosphate-buffered saline (PBS) containing 1% Triton X-100 at a room temperature, for 30 min. After blocking with PBS containing 2.5% bovine serum albumin (Sigma-Aldrich) and 0.2% Triton X-100 for 30 min, the organoids were incubated with the anti-ER antibody (Roche Diagnostics) at 4℃ for 48 h. Subsequently, they were incubated with secondary antibodies conjugated with Alexa-555 (Thermo Fisher Scientific) and Hoechst33342 (Thermo Fisher Scientific) at 4℃ for 72 h. Finally, the organoids were mounted with SCALEVIEW-S4 (Fujifilm Wako Pure Chemical Corporation), and fluorescence images were obtained using a Leica TCS SPE confocal microscope (Leica Microsystems, Wetzlar, Germany).

Evaluation of the ER-, PGR-, and Ki67-positive rate in organoids

After performing IHC, the number of all nuclei as well as the ER-, the PGR-, and the Ki67-positive nuclei of each organoid were counted to calculate the ER-, PGR-, and Ki67-positive rates of each organoid. For the analysis of the wholemount staining, the number of the nuclei was counted using the images at the maximum diameter of each organoid.

Transcriptome analysis

A Homo sapiens whole transcriptome sequencing was performed to examine the different gene expression profiles. Total RNA was extracted with the use of RNeasy mini (QIAGEN, Germantown, MA) from the paired primary and passaged organoids of KAN34 and KAN37. Due to the limitation imposed by the amount of primary organoids, a single sample was analyzed for each line. QC, library construction, and transcriptome analysis through RNA sequencing were conducted by Macrogen Japan, Tokyo. The RNA amplification was performed by using the SMART Seq V4 Ultra Low Input RNA Kit (Takara Bio, Kusatsu, Japan) and by following the manufacturer’s protocol. The TruSeq Stranded mRNA Library Preparation v2 kit (Illumina, San Diego, USA) was used for the library construction, and the NovaSeq 6000 (Illumina) was used for RNA sequencing. All processes from the point of the alignment to the posttreatment of the FASTQ data obtained by RNA sequencing were performed by using the following programs: FastQC 0.11.7, Trimmomatic 0.38, HISAT2 2.1.0, Bowtie 2.3.4.1, and StringTIe 2.1.3b.

Real-time polymerase chain reaction (RT-PCR)

Total RNA was extracted from organoids using a RNeasy Mini Kit (QIAGEN). RT-PCR reactions were performed as described previously [15], and the primer sequences used are shown in Table S2.

Western blotting

Western blotting was performed as previously described [15]. The primary antibodies against the cleaved NOTCH1 (Val1744) (D3B8) and β-actin (AC-74) were obtained from Cell Signaling Technology and Sigma-Aldrich, respectively.

Statistical analysis

All statistical analyses were performed by using GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). The statistical significance was tested through a Student’s t-test in the case of single comparisons or through a one-way analysis of variance followed by a Tukey’s test in the case of multiple comparisons. A p-value lower than 0.05 was considered as statistically significant.

Preparation and culture of patient-derived breast cancer organoids

We obtained 39 tumor specimens from 37 breast cancer patients. These breast cancer specimens were of the following subtypes: 33 luminal, two human epidermal growth factor receptor 2 (HER2)-enriched, one triple-negative, and one ductal carcinoma in situ (subtype not assessed) (Table S1). The specimens’ sizes were approximately 0.3 × 0.3 × 1.5 cm in the case of the biopsy-derived ones and 0.4 × 0.4 × 1.0 cm in the case of the surgery-derived ones; these dimensions differed due to the different sizes of the needles used (Fig. 1A). Two thirds of each specimen were subjected to the organoid preparation, whereas the remaining one third was fixed with formalin for tissue analysis. Organoids were prepared as clusters of the cancer cells (Fig. 1B), and were cultured according to a previously reported method [9]. Organoids were successfully prepared from all of the breast cancer tissues (39/39; 100%); of these, all but one (38/39: 97%) were grown, and 35 were passaged for more than five times (35/39; 90%) (Table S1). Thus, the organoids obtained from a relatively small size biopsy samples of breast cancer tissues were successfully prepared, cultured, and passaged in our study.

Primary Luminal-type Breast Cancer Organoids Maintained The Properties Of The Patient Tumors

In this study, we focused on the luminal subtypes of breast cancer. We investigated the morphology of the primary luminal breast cancer organoids that were prepared directly from the patients’ tissues (and were not passaged in vitro at all), and we compared it with that of the corresponding patient tumors. In all four cases examined, the hematoxylin and eosin staining revealed a similar cell morphology in terms of the nuclear/cytoplasmic ratio as well as the nuclear morphology (Fig. 1C). Furthermore, the ER-, PGR-, Ki67-, and CK19-positive rate were consistent with those of the patient specimens (Fig. 1D). Moreover, we quantified the ER-positive rate of individual organoids in each organoid pool deriving from 12 different patient tumors (Fig. S1). The ER-positive rate of the patient tumors was generally well maintained in these primary organoids, although in some cases the ER-positive cells decreased within some organoids, thereby exhibiting a wide range of ER-positive rates. Taken together, the primary luminal breast cancer organoids maintained the histological properties of the patient tumors of origin.

Er Expression In The Organoids Decreased From Passage To Passage

Subsequently, we examined the ER-positive rates after passaging for more than five times. In the patient-derived organoids generated from the 32 luminal-type breast cancer samples and a single HER2-enriched-type breast cancer sample, the average rate of the ER-positive organoid cells generally decreased after passaging (30/33; 91%; Fig. 2A). When assessed at the individual organoid level, the ER-positive rate was found to be diminished to varying degrees (Fig. 2B). The ER-positive rate remained high in only three organoids (namely, KAN14, KAN20, and KAN38); hereafter referred to as “ER maintainers” (3/33; 9%; Figs. 2B and 2C). In three different organoids (namely, KAN8, KAN17, and KAN34), the ER expression was completely lost; hereafter referred to as “ER losers” (3/33; 9%; Figs. 2B and 2C). However, in most organoids, the ER expression was found diminished yet still detectable to some extent; hereafter referred to as “ER diminishers” (27/33; 82%; Figs. 2B and 2C). In contrast, the Ki67-positive rates were generally maintained after passaging, although some cases exhibited an increase or a decrease (Fig. S2). We further investigated the passage-by-passage changes in the positive rates of ER. In the ER maintainers, the ER expression was retained at high levels throughout the passages studied (Figs. 3A and S3). The ER losers suddenly lost their ER expression during passage #2 in both KAN17 and KAN34 (Figs. 3A and S3). On the contrary, the ER diminishers exhibited various pattern of ER-positive rate decrease (Figs. 3A and S3). The ER expression gradually decreased in KAN15, decreased during passage #1 and was thereafter kept at low levels in KAN32, or decreased during passage #1 and was gradually recovered in KAN31 and KAN33. These findings indicate that the ER-positive rate generally decreased, but the pattern of the decrease depended on the organoid line.

Transition from a luminal- to a basal-type during the in vitro passaging of the organoids

Since the ER expression decreased after passaging in most of the organoids studied, we further investigated their differentiation and proliferation status. In an ER maintainer organoid line (KAN20), the expression of PGR was retained at high levels throughout the passages (Fig. 3B), CK8 (a luminal marker) was ubiquitously expressed and remained at these levels even after passage #5, whereas CK5 (a basal marker) remained barely expressed (Fig. 3C). In an ER loser organoid line (KAN17), PGR suddenly disappeared after passage #2 simultaneously with the ER loss (Fig. 3B), whereas the CK8-single-positive cells (that were dominant in the primary organoids) disappeared and completely changed into CK5-single-positive cells by passage #12 (Fig. 3C). In an ER diminisher organoid line (KAN15), PGR also suddenly disappeared after passage #2 (Fig. 3B). The primary organoids composed exclusively of CK8-single-positive cells (Fig. 3C), or of mixtures of CK5-only- or CK8-only- and CK5/8-double-positive cells (Fig. 3C), whereas the pure CK8-single-positive organoids disappeared and the CK5-single-positive or CK5/8-double-positive cells remained after passage #5 (Fig. 3C).

In terms of their proliferation, the Ki67-positive rates were once reduced after the preparation of the primary organoids in all the cases (Fig. 3D). In the case of KAN20, after the first passage, the organoids exhibited similar levels of Ki67-positive rate to those of the patient tumor of origin (Fig. 3D). In the case of KAN17, the Ki67-positive rates increased after passage #2, and gradually decreased with further passaging (Fig. 3D), whereas in the case of KAN15, they gradually increased (Fig. 3D). Thus, through passaging, most of the breast cancer organoid cells have shifted (to varying degrees) toward basal-like cells.

The culture method that we employed in this study for the culturing of breast cancer organoids has been reported to be suitable for the culturing of normal mammary gland organoids as well [16]. To compare the features of the organoids obtained from breast cancer samples with those of organoids obtained from normal breast tissues, we have prepared organoids from noncancerous breast tissues that were distant from the tumors of four patients who received mastectomy (KAN31−KAN34). The normal organoids at passage #4 exhibited little variation in terms of their ER-, PGR- and Ki67-positive rates (Figs. S4A−S4C), which is apparently different from the variation exhibited by the corresponding breast cancer organoids (Figs. 2B and S2). Based on their distinct characteristics, the cancer organoids are not likely to be replaced by the organoids of the normal breast cells.

Gene expression profiling revealed the breast cancer organoid cell type transition from the luminal- to the basal-type during passaging

To assess the change of the gene expression during the organoid passaging, we performed an RNA sequencing of the primary and the passaged organoids in two organoid lines (namely, KAN34 and KAN37). The gene expression remained the same for approximately 80% of the genes in both lines. In contrast, the gene expression of the luminal-related genes [17, 18] was found to be decreased after passaging, whereas the gene expression of the basal ones [17, 18] increased (Fig. 4A and 4B). In addition, the gene expression of the stemness-related genes [19–21] was found to be increased after passaging (Fig. 4C). The differences were greater in KAN34 (an ER loser organoid line) than in KAN37 (an ER diminisher organoid line). These findings suggest that the luminal-type breast cancer cells become basal-type ones after several passages in vitro.

Adding a NOTCH inhibitor to the culture medium partially restored the ER expression status in the organoids

Subsequently, we investigated the molecular mechanisms underlying the transition of the differentiation status in breast cancer organoids during an in vitro culturing and passaging. We investigated the NOTCH signaling, since the RNA sequencing analysis suggested that the NOTCH signaling was activated in the passaged organoids (Fig. S5). In addition, it has been reported that the inhibition of the NOTCH signaling can increase the ER expression in ER-negative breast cancer cell lines [22]. Therefore, we examined whether the ER expression could be restored by the addition of a NOTCH inhibitor (such as DAPT) in the culture medium. After passaging three different organoid lines—KAN17 (an ER loser), KAN30 (an ER diminisher), and KAN41 (an ER diminisher)—we replaced the culture medium with a DAPT-containing one, we cultured the organoids for 7 days, and we analyzed their gene expression and differentiation status. The expression levels of HES1 (hairy and enhancer of split-1; a target gene of NOTCH signaling) were found to be suppressed by DAPT in all three lines (Fig. 5A), whereas the expression levels of some other NOTCH target genes (such as HEY1, CDKNA1, and NOTCH1) were found to be decreased in some lines (Fig. 5A). The ER-positive rate was found to be significantly increased in all the lines (Fig. 5B), accompanied by an increase of the CK8- and a decrease of the CK5-positive cells (Fig. 5C). The change was less evident in the ER loser organoid line (KAN17). Thus, the inhibition of the NOTCH signaling was able to restore (to some extent) the altered differentiation of the breast cancer organoids.

ER expression in luminal-type breast cancer organoids was preserved through the inhibition of NOTCH signaling only at passaging

Since the ER expression suddenly disappeared after passaging in some of the organoids (Figs. 3A and S3), we speculated that the passaging itself (rather than the culturing) has triggered the alteration of the differentiation status of the luminal-type breast cancer cells. Indeed, the cleavage of NOTCH to its active form, the NOTCH intracellular domain (NICD), was clearly detected after the passaging of the breast cancer organoids, and it was inhibited by the pretreatment with DAPT (Fig. 6A). HES1 was found to be induced immediately after passaging, and to recover to basal levels afterwards (Figs. 6B and 6C). Other NOTCH target genes (such as HEY1, CDKNA1, and NOTCH1) were also mostly induced by passaging, and were blocked by DAPT in all of the organoids examined (Fig. 6C). In contrast, ESR1 (the ER-encoding gene) neither increased as a result of passaging (except for KAN37 and KAN41), nor decreased as a result of the DAPT pretreatment (except for KAN20), thereby suggesting that the ESR1 is not directly suppressed by the NOTCH signaling (Fig. S6). The activation of the NOTCH signaling by passaging was further confirmed by an in situ hybridization for HES1 (Fig. S7). We, then, examined whether the blocking of the NOTCH signaling only at the time of the passaging can rescue the downregulation of the ER in primary luminal-type breast cancer organoids. The NOTCH signaling was blocked by DAPT only before and after the passaging. After several (3−5) passages, the ER expression levels were found to be significantly higher in the DAPT-treated organoids than in the DMSO-treated controls in three ER diminisher organoid lines, whereas they were almost completely maintained in the case of the KAN39 organoids after passage #3 (Figs. 6D and 6E), along with an increase of CK8- and a decrease of CK5-positive cells (Fig. 6E). Taken together, the disruption of the organoids during their passaging seems to trigger the NOTCH signaling, that plays a critical role in altering the differentiation status of the organoids.

We have succeeded in generating organoids from luminal-type breast cancer samples with a high success rate. Since the long-term culturing of breast cancer organoids was first reported [9], there have been several reports on breast cancer organoids by using this method [11, 23–25]. Although there is no clear definition of a “successful organoid culture,” the efficiency of generating organoids from ER-positive breast cancers ranges from 50–70% in these reports. We were able to generate organoids with a 100% success rate from small pieces of luminal-type breast cancers, and expanded them for more than five passages in the case of 34 of these lines. One reason for the high efficiency recorded in this study is the adoption of the modified CTOS method [14], in which the organoids are prepared and cultured as clusters, thereby maintaining their cell-cell contact throughout the process of preparation and culturing; a fact that might have contributed to the reduction of the incidence of cell death, and improved the recovery rate (as compared to the method involving the dissociating of the tissues into single cells). These improvements will be critical for the undertaking of clinical tests by using primary organoids.

The decrease of the ER expression in cultured organoids has been previously reported by several researchers [9–12]. In these reports, the ER expression was histologically judged as “positive or negative” [12] or the ER-positive rate was assessed in all the cells of multiple organoids [9–11]. In this study, we calculated the ER-positive rates of individual organoids, and followed their progress over time, thereby demonstrating an inter- and an intra-organoid heterogeneity in both the extent and the mode of the ER expression reduction. The luminal-type properties were well preserved in primary cultures and in a minor subgroup of the ER maintainer organoid lines. In contrast, the properties completely shifted to basal-like types in the ER loser organoid lines. These organoids were clearly different from those generated from normal breast tissues that consisted of variously differentiated cells (Fig. S4D), as previously reported [16]. Most of the generated organoid lines were found to be ER diminishers; the cells of these lines obtained basal-like characters to varying degrees. Some of the ER diminishers almost completely lost their ER expression (Fig. 2B), whereas the passaged KAN40 organoids consisted of pure CK8-expressing cells and their ER-positive rates were substantially decreased (Figs. 6D and 6E). Thus, the potential of changing the differentiation status in vitro highly depends on each organoid line. The assessment of the differentiation status is critical when the passaged organoids are used for the study of luminal-type breast cancer. Our experiments also revealed that the inhibition of the NOTCH signaling was able to restore the diminished ER expression. By using primary organoids, or passaged organoids that can either maintain or can restore their luminal-type cell characters through NOTCH inhibition, one can develop a novel in vitro platform for the study of luminal-type breast cancer.

Through gene expression profiling and immunohistochemistry, we revealed that the passaged organoids changed their cell status from a luminal- to a basal-like one. The dominant expansion of specific subclones or the epithelial-mesenchymal transition (EMT) have been speculated as the causes of the reduced ER expression in organoid cultures [11], although a clear mechanism has not yet been elucidated. Since the organoids in this study abruptly decreased their ER expression and that of other luminal markers after the first or the second passage (in most of the cases), their replacement is unlikely due to the predominant growth of ER-negative subclones. In terms of EMT, although some genes (such as SNAI2 and VIM in KAN34, and TWIST1 in KAN37) were found to be upregulated in the passaged organoids, most of the EMT-related genes did not change (Fig. S5), thereby suggesting the absence of a typical EMT. In contrast, the observed decrease of the expression of luminal markers and the emergence of stemness markers suggest a de-differentiation to the stem-like status. We have previously reported that the disruption and the remodeling of 3D structures in colorectal cancer organoids can promote stemness [26]. Moreover, the culture method used in this study is a stem cell culture method [27], and as a result, the de-differentiation to a stem cell-like status is not surprising. Taken together, although the possibility of a direct conversion to a basal-like status (trans-differentiation) cannot be ruled out, the de-differentiation to a stem-like status and the redifferentiation to a basal-like lineage is more likely. The ER maintainer organoid lines may be less prone to be de-differentiated, and both the ER maintainer and the ER loser organoid lines may be more committed to their respective lineages.

The activation of NOTCH by passaging was blocked by a γ-secretase inhibitor, although the molecular mechanisms underlying the passaging-induced activation of the NOTCH signaling remain unclear. The passaging procedure in this study consists of an enzymatic digestion and a mechanical dissociation. Treatment with EDTA or trypsin can reportedly activate the NOTCH signaling [28, 29], whereas the mechanical dissociation might be able to activate the upstream proteases. In any way, we did not disassociate the organoids into single cells during passaging, and the undertaken in situ hybridization suggested that the extent of the HES1 expression was quite heterogenous. Therefore, it is not likely that an ubiquitous activation of NOTCH signaling occurred in all of the cells at the same time. The change of the differentiation status might be propagated in the clusters through the NOTCH signaling or through other pathways originating from the NOTCH-activated cells. The reason why the ER expression of the primary organoids were fairly maintained in this study might be due to the method employed for the organoid preparation from patient tumors, in which the cancer cell clusters were retrieved from the patient tumors as intactly as possible, and therefore the NOTCH activation might have been minimal.

The alteration of the ER-positive rate during the progression or the treatment of the luminal-type breast cancer is not a rare event in clinic [30, 31]. Aitken et al. [30] have reported that in 31.8% of 110 cases, the ER-positive breast cancer changed into ER-negative in metastatic lymph nodes. Lindstrom et al. [31] have reported that in 34.3% of 329 cases, the ER status changed from positive to negative in local and systemic relapse cases with poorer prognosis. In our study, the complete loss of the ER occurred in 9% (3/33) of our organoid lines. The negative conversion of ER and its recovery through the NOTCH inhibition has been previously reported by a pioneering study using breast cancer cell lines such as MCF7 and T47D [22]. These cell lines maintain quite a stable differentiation status in vitro, and their luminobasal cells appeared only after a xenograft formation. Moreover, their luminobasal cells expanded only after entering a crisis and resuming after 30−40 days of culturing following an exposure to estrogen withdrawal conditions. In contrast, we have herein demonstrated the highly changeable potential of the lumina-type breast cancer organoids.

We prepared cancer organoids from the breast cancer lesions of patients and achieved a high success rate using the CTOS method. Our findings suggest that the differentiation status of luminal-type cancer cells is flexible in vitro. Moreover, by inhibiting NOTCH signaling we can preserve the differentiation status of luminal-type breast cancer organoids. Given that alteration of the differentiation status in breast cancer is one of the causes of drug resistance, breast cancer organoids might be useful for studying the mechanisms underlying such alterations.

3D: Three-dimensional; CDK4/6: Cyclin-dependent kinase 4/6; CDKN1A: Cyclin dependent kinase inhibitor 1A; CK19: Cytokeratin 19; CK5: Cytokeratin 5; CK8: Cytokeratin 8; CTOS: Cancer tissue-originated spheroid; DAPI: 4',6-diamidino-2-phenylindole; DAPT: N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester; DMSO: Dimethyl sulfoxide; EDTA: Ethylenediaminetetraacetic acid; EGF: Epidermal growth factor; EMT: Epithelial-mesenchymal transition; ER: Estrogen receptor; ESR1: Estrogen receptor 1; FGF10: Fibroblast Growth Factor 10; FGF7: Fibroblast growth factor 7; HE: Hematoxylin and eosin; HER2: Human epidermal growth factor receptor 2; HES1: Hairy and enhancer of split 1; HEY1: Hairy/enhancer-of-split related with YRPW motif protein 1; IHC: Immunohistochemistry; mRNA: Messenger RNA; NICD: Notch intracellular domain; NOTCH1: Neurogenic locus notch homolog protein neurogenic locus notch homolog protein 1; PGR: Progesterone receptor; QC: Quality control; RT-PCR: Real-time polymerase chain reaction; SNAI2: Snail homolog 2; TPM: Transcripts per million; TWIST1: Twist-related protein 1; VIM: Vimentin.

Acknowledgements

We thank Mr. Hiromitsu Tazawa (Clinical Bio-resource Center of Kyoto University’s Hospital) for his administrative support and Mitsuaki Ishida for his support in the pathological analysis.

Author contributions

MM, YM, TS, and MI designed the study. HU and CS performed the experiments. JK and RO supervised the experiments. YK, MT, and TS acquired the clinical samples and data. HU, RO, and MI conducted the data analysis. HU and MI wrote the manuscript. HU, JK, KO, RO, and TS reviewed and revised the manuscript. All authors read and approved the final manuscript.

Funding

This research was supported by a collaboration grant from Kyoto University-KBBM (H.U., C.S., J.K., Y.M., M.M., T.S., M.I,) and the Takeda Science Foundation (M.I.).

Availability of data and materials

The datasets generated and analyzed during the current study are available in DDBJ (accession number: DRR412393-DRR412396).

Ethics approval and consent to participate

This study was approved by the institutional ethics committees of Kyoto University (R2220, R2444) and Kansai Medical University (2019245, 2021090). Written informed consent was obtained from participants. This study was performed in accordance with the Declaration of Helsinki.

Consent for publication

Written consent for publication was obtained from participants.

Competing interests

J.K., K.O., and M.I. are members of the Department of Clinical Bio-resource Research and Development at Kyoto University, which is sponsored by KBBM, Inc. H.U. and C.S. are employees of KBBM Inc. The other authors declare no conflict of interest. This work was supported in part by the collaboration grant from KBBM- Kyoto University.

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Competing interest reported. J.K., K.O., and M.I. are members of the Department of Clinical Bio-resource Research and Development at Kyoto University, which is sponsored by KBBM, Inc. H.U. and C.S. are employees of KBBM Inc. The other authors declare no conflict of interest. This work was supported in part by the collaboration grant from KBBM-Kyoto University.

SupplFigures.pdf
Figure S1 Quantitative analysis of the ER-positive rate in primary organoids After immunostaining the paraffin sections of the organoids for ER, the ER-positive and -negative cells were counted in individual organoids. The ratio of positive nuclei to total nuclei is shown. Each dot represents an individual organoid. Each red line indicates the ER-positive rate of the pathological diagnosis for each of the patient tumor samples. Figure S2 Quantitative analysis of the Ki67-positive rate in passaged organoids After immunostaining the paraffin sections of the organoids for Ki67, the Ki67-positive and -negative cells were counted in individual organoids. The ratio of positive nuclei to total nuclei is shown. Each dot represents an individual organoid. Each red line indicates the Ki67-positive rate of the pathological diagnosis for each of the patient tumor samples. Sample IDs are shown in three colors: ER maintainers (red), ER diminishers (green), and ER losers (blue). Figure S3 Quantitative analysis of the ER-positive rate in the organoids at each passage Each dot represents an individual organoid. Each red line indicates the ER-positive rate of the pathological diagnosis for each of the patient tumor samples. Figure S4 Differential status of normal breast organoids after passaging (A-C) Quantitative analysis of the ER- (A), PGR- (B), and Ki67- (C) positive rates in individual organoids deriving from normal parts of the indicated resected breast tissues at passage #4. Each dot represents an individual organoid. (D) Representative images of the CK5/8-double-staining of the KAN32 and KAN33 organoids shown in (A-C). Scale bar: 50 μm. Figure S5 Expression of NOTCH-signaling- and EMT-related genes in primary and passaged organoids Relative expression of NOTCH-signaling- and EMT-related genes obtained from the RNA sequencing analysis presented in Figure 4. Fold-change of the TPM is shown. Figure S6 ESR1expression before and after passaging Semi-quantitative RT-PCR for ESR1. The organoids and the conditions are indicated. Symbols used: *, p < 0.05; **, p < 0.01; ****, p < 0.0001. Figure S7 In situ hybridization for the assessment of HES1 In situ hybridization was performed by using the RNAscope 2.5 HD Reagent Kit - BROWN (322300; Advanced Cell Diagnostics, CA, USA) and the RNAscope Probe - Hs-HES1 (311191; Advanced Cell Diagnostics, CA, USA) according to the manufacturer’s protocol. The ratios of the HES1staining positive area to the total area of nuclei were calculated by using the ImageJ (Fiji) software (https://imagej.net/software/fiji). (A) In situhybridization of HES1 for two ER diminisher organoids (KAN30 and KAN41). The organoids were fixed before and immediately after the passaging in the presence or absence of a pretreatment with DAPT. Scale bar: 50 μm. (B) Quantitative analysis of (A). The ratios of the staining positive area to the total area of the nuclei were calculated. Symbols used: ns, nonsignificant; **, p < 0.01; ***, p < 0.001.
TableS1.xlsx
Table S1 Clinicopathological information of the patients whose specimens were used in the current study
TableS2.xlsx
Table S2 Primer sequences used for the undertaking of RT-PCR

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The differentiation status of luminal-type breast cancer–derived organoids can be altered in vitro through the activation of NOTCH signaling

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Background

Methods

Statistical analysis

Results

Preparation and culture of patient-derived breast cancer organoids

Primary Luminal-type Breast Cancer Organoids Maintained The Properties Of The Patient Tumors

Er Expression In The Organoids Decreased From Passage To Passage

Discussion

Conclusions

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