Introduction
Solid tumors are heterogeneous in their cell composition, with a small subset of tumor cells sharing certain biological and molecular properties with tissue-specific stem cells (SC) [
1‐
3]. This cell population has long-term renewal potential, supporting a proliferation hierarchy among cancer cells. In some tumors, the genes and signaling pathways that regulate normal SC roles also function as oncogenes or regulate tumor maintenance and progression [
4‐
16]. One of the best-characterized examples is WNT–β-catenin signaling, which is essential for the maintenance and proliferation of SCs [
17]. Importantly, this pathway is frequently mutated in colorectal cancers [
18] and is required to sustain tumor growth and progression in several different types of cancers, including colorectal cancer, leukemia and skin basal cell carcinoma [
19]. Consistent with the reprogramming of tumor cells into an embryonic-like fate, similarities between embryonic mammary SCs and the basal-like and HER2-positive breast cancer subtypes (which are less differentiated than other breast cancer subtypes) have also been described [
20]. All those observations are consistent with the idea that, for tumor initiation, adult cells are required to undergo reprogramming to a progenitor-like fate.
Many of the current therapeutic strategies aimed at eliminating cancer cells involve treatment with standard antiproliferative chemotherapy, which often has limited benefits. The residual population of chemotherapy-resistant tumor cells capable of regenerating the disease is—at least by definition—enriched in cancer stem cells (CSCs) [
1]. This fact has inspired the design of numerous antitumor therapies directly targeting the CSC niche, based on inducing their terminal differentiation. Indeed, the original idea of anti-CSC therapy arose in the 1970s and 1980s, from the observation that leukemic cells are blocked in an undifferentiated state. The use of all-trans retinoic acid induced terminal differentiation of leukemic cells [
21]—and currently is the standard of care for the treatment of patients with acute promyelocytic leukemia. The success of all-trans retinoic acid therapy inspired other therapies that were based on inhibiting epigenetic regulators to induce cancer differentiation in multiple hematological malignancies [
22] and the same mechanism also shows certain promise in solid tumors. Nevertheless, it is now established that even differentiated cells can be reprogrammed into stem-like cells, suggesting that cell state reprogramming is more common and occurs in more diverse cell types than previously thought [
23,
24]. Indeed, this type of reprogramming can be used to re-establish stem-like hierarchies in tumors even after elimination of putative CSCs [
25]. Therefore, eliminating unstable cells and also abrogating the mechanisms by which tumor cells gain cell state plasticity may be the most productive differentiation strategy. Such complexity makes the enticing therapeutic targeting of undifferentiated cancer cells still uncertain.
Novel players in carcinogenesis are microRNAs (miRNAs), which are epigenetically regulated but also control epigenetic events [
26]. miRNAs comprise a class of small noncoding RNAs involved in posttranscriptional regulation of gene expression. miRNAs act by inhibiting translation of target mRNAs, and it is estimated that one-third of protein-coding mRNAs are subjected to regulation by miRNA. miRNA deregulation has been implicated in cancer development, and both oncogenic and tumor-suppressor miRNAs have been identified, many of which act through inhibition of translation of proteins controlling cell proliferation, survival and development [
26].
Fuchs’ laboratory first described an in vivo role for microRNA 203 as a suppressor of stemness in developing epidermis [
27]. Soon after that, we described miR-203 as a tumor suppressor in hematopoietic tumors. Our laboratory found that miR-203 expression was frequently silenced in mouse and human T and B cell malignancies through hypermethylation of its genomic region, and ABL1 and BCR-ABL1 fusion transcripts are indeed direct targets of miR-203-mediated translational repression [
28]. The same year, a landmark paper from Massague’s group identified a set of eight microRNAs whose expression was inversely correlated with the metastatic potential of human breast cancer cell lines [
29]. Though not studied further, miR-203 was among the eight miRNAs initially identified in that study. In the years since this report was published, miR-203 has been shown to regulate genes involved in crucial tumor pathways, such as signal transduction (BCR-ABL1), stemness (p63, BMI1), migration (LASP1, ASAP1), as well as known regulators of metastasis (SNAI1/2) among many others [
30‐
39]. However, the capacity of miR-203 to fine-tune cancer cell differentiation remains uncertain and deserves a more focused research.
Recently, our laboratory has identified an unprecedented role of miR-203 modulating both reprogramming from somatic to pluripotent cells [
40] and the differentiation capacity of stem cells [
41]. Our data support the intriguing fact that a brief exposure to miR-203 blocks reprogramming to pluripotency while expanding the differentiation efficiency of stem cells. Such effects are mediated by direct or indirect targeting of the epigenetic landscape, making pluripotent cells more proficient for subsequent differentiation.
Given the obvious parallelisms between tumorigenesis and pluripotency [
42‐
44], we evaluated the outcomes of miR-203 treatment on cancer cell differentiation. Using the classical MMTV-PyMT transgenic mice as a breast cancer model [
45], we demonstrate here that miR-203-mediated effects on cellular reprogramming and cell differentiation can be advantageous in antitumor therapy. Combining in vivo approaches and their direct version on in vitro settings by tumor-derived organoids, we show that a brief exposure to miR-203 controls the self-renewal and proliferative capacity of breast cancer cells, attenuates migratory abilities and provokes a switch from a basal tumor phenotype to a more differentiated luminal-like status, similar to that observed in non-tumor cells.
Discussion
Cancer has been broadly interpreted as a caricature of normal tissue development. Cellular programs regulating tissue plasticity, self-renewal and expansion are exquisitely orchestrated under physiological conditions. However, aberrant tumor mechanisms unbalance this coordinated cell plasticity and give rise to immature or dedifferentiated tumor cells. Indeed, it is now widely accepted that, for tumor initiation, adult cells experience reprogramming to a progenitor-like fate [
67]. Thus, tumor dedifferentiation supports cancer progression, relapse and metastasis. Traditional chemo- and radiotherapy generally involves the elimination of proliferating tumor cells. Instead, the differentiation therapies offer the possibility of coaxing cancer cells into becoming normal cells, reactivating the endogenous differentiation programs to resume maturation. Cancer differentiation approaches are still evolving and require novel methodologies to reach efficient therapies. Trusting this general believe, we decided to examine the antitumor effects of miR-203 from a cancer differentiation perspective. This microRNA has been recently described by our group to fine-tune the critical balance between reprogramming, stemness and differentiation programs: miR-203 blocks somatic-to-pluripotency reprogramming [
40], while potentiates differentiation of stem cells to a mature and terminal state [
41]. We hypothesized that such effects could be applied to cancer differentiation and therefore would point to miR-203 as a promising tool for differentiation-based antitumor therapy.
In vivo, we tested different treatment schedules, trying to understand the consequences of treatment intermittency. Interestingly, only when mice were exposed to miR-203 from tumor onset to the end of the experiment, tumor initiation and growth were completely prevented. When miR-203 treatment was intermittent, we noticed a significant control on tumor growth and a considerable delay in tumor initiation, while we did not completely avoid the latter. In this line, recent advances on stem cell biology have demonstrated that stem cell plasticity represents one of the major therapeutic challenges for differentiation therapies. Several studies have provided evidence that both CSCs and non-CSCs are plastic and capable of undergoing phenotypic transitions in response to appropriate stimuli. This notion was for instance exemplified by a study in which cell populations displaying stem cell-, basal- or luminal-like phenotypes were isolated from breast cancer cell lines [
68]. In vitro, all three subpopulations were able to generate cells of the other two phenotypes. This phenotypic inter-conversion was stochastic and not determined by the cell phenotype of origin. Thus, it is accepted now that CSC and non-CSC states are not hardwired: considering that plasticity may be in tumor cells as extensive as it is in healthy tissues, CSCs would be always recreated. This fact could explain the successful outcome when miR-203 treatment was uninterrupted (Fig.
2), theoretically capturing any newly formed CSC. However, the intermittency in miR-203 exposure would tentatively allow the undifferentiated population to restore, favoring then the tumor initiation process. We interpreted that miR-203 exposure was able to maintain the stem-like capacity delimited and therefore tumor growth under control in any regimen tested. One of the key results in vivo, denoting the strong effect of miR-203 on tumor differentiation, was the lack of metastasis (suggestive of detrimental migratory and invasive capacity) detected on those mice exposed to miR-203 in any of the treatment schedules tested, while their control counterparts experience a metastasis incidence of 31%. Of interest, in vitro cell cultures derived from patient biopsies exhibited a polarized and migratory cell population that was completely abolished by miR-203 treatment. Together, those two interesting observations prompt us to speculate that miR-203 impact on cancer cell differentiation plays a role in invasion and metastasis.
Inspired by the natural development of the mammary epithelium, Hans Clevers and collaborators established culture protocols that allow the generation and long-term expansion of three-dimensional mammary gland- or tumor-derived organoids [
69,
70]. We efficiently produced organoids from the mouse tumors or mammary glands, recapitulating the tridimensional architecture and the molecular features of the source tissue, and thus allowing a deeper analysis of the cancer stem cell behavior in vitro.
Our first striking observation was the remarkable differences on organoid morphology upon miR-203 treatment: The structure of control tumor-derived organoids was notably compacted, disorganized, dense and in most cases, grape-shaped, while the miR-203-treated tumor-derived organoids were predominantly cystic, suggesting an epithelial luminal origin. Of interest, the cystic morphology was reproduced any time the tumor-derived organoids were yielded to transient miR-203 treatment in vitro.
This observation is not surprising if we consider that the adult virgin mammary gland is a highly organized tree-like structure, formed by ducts with hollowed lumen. It has been widely demonstrated that a controlled and delimited induction of apoptosis is crucial for clearing the lumen in terminal end buds during puberty. Apparently, the differentiation process observed in the organoids implies a similar process, spatially and temporally organized, where the lumen is shaped and the cells distribute adjacent the newly formed cystic structure. Moreover, those cystic organoids resulting from miR-203 exposure, either in vivo or in vitro, collapsed after a few passages, contrary to the control organoids never treated with the microRNA, which were long-term maintained in culture, as published before [
54,
69,
70]. The short lifespan of organoids exposed to miR-203 clearly pointed to an exhaustion of the self-renewal capacity of the culture—possibly accompanied by an increased cell death index—and implied a direct detrimental effect by this miRNA on the propagation and expansion of the organoids. The induction of differentiation comprises a plethora of signals, cellular-transduced and ultimately translated into a complex combination of responses. Accordingly, Ki67, CK5, CK14 and ALDH1/2 levels were diminished in those miR-203-exposed organoids, while markers such as CK8/18 were induced, implying a shift from dedifferentiated basal to a more differentiated luminal-like phenotype.
One of the questions we have not answered in this work is the cellular origin of miR-203. Our data in organoids suggest a cell-autonomous effect on mammary gland cells, and indeed denote a clear luminal character. It has been published that miR-203 is activated during luminal epithelial differentiation and this pattern is observed in the murine mammary hierarchy [
71]. Also, in a paper where microRNAs signatures of distinct mammary epithelial cell types were analyzed, miR-203 was found as delimited to luminal cells again, targeting basal-restricted genes [
72]. Although a deeper analysis should be done to elucidate the cellular subpopulation responsible for such observed effects, it is tempting to speculate that-at least in our model system- the over-expression of miR-203 in luminal mammary gland cells exacerbates a naturally designed differentiation program in this cell subtype.
Of interest, the healthy mammary gland tissue-derived organoids exhibited a very similar phenotype to the one observed in the miR-203-treated tumor organoids (Figs.
5D, 5E,
6C, D and Additional file
1: Fig. S2B, S2C). Several markers associated to differentiation were found to be more expressed in healthy tissue-derived as well as in miR-203-exposed tumor organoids.
Deepening into the differentiation concept, we tested in our organoid platform some previously defined differentiation conditions, such as FGF2-mediated induction of mammary branching or a culture medium described for mammary epithelial cell differentiation. The tumor-derived organoids were mostly condensed and grape-shaped upon basic expansion media, and shifted to a predominantly hollow cysts morphology when exposed to the epithelial differentiation media or (even more dramatically) to miR-203, suggesting that any of those treatments were boosting the cyst-forming ability of mammary epithelial cells. As expected, FGF2 treatment always induced the mammary trees typical of branching morphogenesis [
56] either on non-tumor or tumor organoids, exposed or not to miR-203. The intriguing fact that, upon FGF2 treatment, miR-203 does not induce epithelial cystic formations but instead favors the branching constructions, was in concordance with our previous works and others [
40,
41,
73] and suggests that, submitted to a strong differentiation scenario, miR-203 acts always as a differentiation enhancer and not as a reprogramming inducer, and also reinforces its role as a regulator of branching morphogenesis and basement remodeling.
When the transcriptomic profiles of those organoids were tested by RNA sequencing, we noticed a remarkable impact of miR-203 treatment on the differentiation of tumor-derived organoids, while the healthy tissue-derived organoids showed very little alterations when exposed to miR-203. These data were in concordance with the phenotypically observed modifications incited by miR-203: Whereas miR-203 exposure completely shifted the shape of tumor organoids, no notable changes were induced on non-tumor organoids. This suggests a fascinating differential impact of this microRNA on tumor and non-tumor tissues that deserves to be further explored. Upcoming works based on single cell analysis would define the mechanistic insights of miR-203 as a tumor differentiation agent, outlining its influence on the transcriptomic, genomic and epigenetic landscapes of the different cell subtypes.
Several targets have been defined for miR-203 in cancer, conferring mostly a role as tumor suppressor [
30], or eventually, as tumor promoter [
74]. This apparent discrepancy could be explained by the distinct models and in vitro systems used in such studies, and how the context strongly influences the microRNA target dependencies and outcomes. Our work highlights how dedifferentiation influences tumorigenesis, and in the context of breast cancer, the potential therapeutic advantages of targeting stem-like basal cells and differentiating them into luminal cells. Thus, the data presented here not only confirm the antitumor effects mediated by miR-203, but also particularly denote its influence on cancer differentiation, both in murine and patient-derived samples. This work undoubtedly opens new perspectives on the potential therapeutic applications of miR-203 in cancer.
Methods
Animal models and procedures
Animal experimentation was performed according to protocols approved by the CNIO-ISCIII and the UCM Ethics Committee for Research and Animal Welfare (CEIyBA) and Madrid Regional Government, according to European official regulations. The miR-203 inducible model was generated by cloning a 482-bp genomic mmu-mir203 sequence into the pBS31 vector for recombination into the ColA1 locus in embryonic stem cells. The resulting knock-in allele [ColA1(miR-203)] was combined with a Rosa26-M2rtTA allele [Rosa26(rtTA)] for doxycycline-dependent induction as described previously [
41]. PyMT [FVB/N‐Tg(MMTV‐PyVT)
634Mul/J] mice were kindly provided by Miguel Quintela (CNIO, Spain). Mice were then crossed to obtain the Tg (MMTV-PyMT); Rosa26(rtTA); and ColA1(miR-203) strain, which has been used throughout this work. To induce miR-203 expression in vivo, doxycycline (Dox) was orally administered to mice in diet (Dox-delayed release pellets, from Jackson laboratories) following the different schedules indicated in Figs.
1,
2,
3. As control, Dox treatment was applied to Tg (MMTV-PyMT); miR-203 (+ / +) mice, which also served as an internal checkup of the Dox treatment itself.
Primers used for genotyping the PyMT transgene were 5′-GGAAGCAAGTACTTCACAAGG-3′ and 3′-GGAAAGTCACTAGGAGCAGGG-5′. Polymerase chain reaction conditions were as follows: 95 °C for 15 min; 94 °C for 30 s; 30 cycles at 59 °C for 45 s; 72 °C for 1 min; 72 °C for 10 min; and then soaking at 4 °C. PCR products are 336 bp (base pair) for wt allele, 438 bp for lox allele, 470 bp for cre allele and 557 bp for PyMT allele. All these animals were maintained in a mixed C57BL6/J × 129 x CD1 genetic background and were housed at the serum pathogen free (SPF) barrier area of the CNIO. Mice were treated in accordance with the Spanish Laws and the Guidelines for Human Endpoints for animals used in Biomedical Research. Mice were observed daily and killed when they showed signs of morbidity or overt tumors.
Micro-computed tomography (micro-CT)
For micro-CT, mice were anesthetized with a continuous flow of 1% to 3% isoflurane/oxygen mixture (2 L/min). Acquisitions were performed using a micro-CT scanner Argus-Vista (SEDECAL, Madrid, Spain) including the whole body in 2-bed position. Tomographic images were reconstructed using a 3D-FBP (filtered back projection) algorithm that produced 55 slices measuring 55 × 55 pixels each. The isotropic resolution of this instrument was 45 µm. The micro-CT image acquisition consisted of 400 projections collected in one full rotation of the gantry in approximately 10 min per bed position. The image acquisition was made without any contrast agent. The X-ray tube settings were 80 kV and 450 µA. For image analysis and quantification, 3D Slicer software was used. Tumor volumes were measured once per week by micro-CT, to determine accurately the tridimensional tumor mass. The investigators were blinded during the entire in vivo experiment. Micro-CT measurements were performed in all cases with no information about the genotype or treatment of every mouse tested. The potential effects of miR-203 on metastasis incidence in the lungs were also analyzed by micro-CT throughout the three in vivo experiments.
Mammary gland-derived organoids culture
Tg (MMTV-PyMT); miR-203 (+
/ +
) and
Tg (MMTV-PyMT); miR-203 (KI/KI) mice (treated or not with Dox in vivo, as indicated in the text) were euthanized, and tumors were extracted. Two random pieces were snap frozen and stored at − 80 °C; two random pieces were fixed in formalin for histopathology and immunohistochemistry analysis and the remainder was processed for the isolation of viable cells. The remaining tissue was minced, washed with 10 mL AdDF + + + (Advanced DMEM/F12 containing 1 × Glutamax, 10 mM HEPES, and antibiotics) and digested in 10 mL BC organoid expansion medium: 10% homemade R-Spondin 1 conditioned medium; 5 nM neuregulin 1 (Peprotech 100-03); 5 ng/mL FGF7 (Peprotech 100-19); 20 ng/mL FGF10 (Peprotech 100-26); 5 ng/mL EGF (Peprotech AF-100-15); 100 ng/mL Noggin (Peprotech 120-10C); 500 nM A83-01 (Tocris 2939); 5 µm Y-27632 (Abmole); 500 nM SB202190 (Sigma S7067); 1X B27 supplement (Gibco 17504-44); 1,25 mM N-Acetylcysteine (Sigma A9165); 5 mM nicotinamide (Sigma N0636); 1X Glutamax (Invitrogen 12634-034); 10 mM HEPES (Invitrogen 15630-056); 100U/mL Penicillin/Streptomycin (Invitrogen 15140-122); Primocin (Invitrogen Ant-pm-1); and Advanced DMEM/F12 (Invitrogen 12634-034), containing 1–2 mg/mL collagenase (Sigma, C9407). Digestion was performed on an orbital shaker at 37 °C for 1–2 h. The digested tissue suspension was sequentially sheared using 10 mL and 5 mL plastic and flamed glass Pasteur pipettes. After every shearing step the suspension was strained over a 100 μm filter with retained tissue pieces entering a subsequent shearing step with ∼10 mL AdDF + + + . 2% FCS were added to the strained suspension before centrifugation at 400 rcf. The pellet was resuspended in 10 mL AdDF + + + and centrifuged again at 400 rcf. In case of a visible red pellet, erythrocytes were lysed in 2 mL red blood cell lysis buffer (Roche, 11814389001) for 5 min at room temperature before the addition of 10 mL AdDF + + + and centrifugation at 400 rcf. The pellet was resuspended in 10 mg/mL cold Cultrex growth factor reduced BME type 2 (Trevigen, 3533-010-02), and 40 μL drops of BME-cell suspension were allowed to solidify on pre-warmed 24-well suspension culture plates (Greiner, M9312) at 37 °C for 20 min. Upon completed gelation, 400 μL of BC organoid expansion medium was added to each well and plates transferred to humidified 37 °C / 5% CO
2 incubators. Medium was changed every 4 days, and organoids were passaged every week: Organoids were resuspended in 2 mL cold AdDF + + + and mechanically sheared through flamed glass Pasteur pipettes. When necessary, very dense organoids were dissociated by resuspension in 2 mL TrypLE Express (Invitrogen, 12605036), incubation for 1–5 min at room temperature, and mechanical shearing through flamed glass Pasteur pipettes. Following the addition of 10 mL AdDF + + + and centrifugation at 300 rcf. or 400 rcf., respectively, organoid fragments were resuspended in cold BME and reseeded as above at ratios (1:1 to 1:6) allowing the formation of new organoids. Single cell suspensions were initially seeded at high density and reseeded at a lower density after ∼1 week. In order to prevent misidentification and/or cross-contamination of BC organoids, we cultured every line physically separate. All organoid lines were frequently tested and resulted in all cases negative in the MycoAlert mycoplasma detection kit (Lonza, LT07-318). For epithelial differentiation, we used the media defined by Lonza (MEGM Mammary Epithelial Cell Growth Medium and Bullekit). Basically, this media has been optimized for the growth of mammary epithelial cells in a serum-free environment and includes BPE, hEGF, insulin, hydrocortisone and GA-1000 (Lonza CC-3150). FGF2 treatment (2 nM; Sigma) was used to induce mammary branching as published before [
56]. For inducing transient miR-203 over-expression, ColA1(miR-203/miR-203); Rosa26(rtTA/rtTA) organoid cultures were treated with Dox (1 µg /mL; Invitrogen) during 5 days. After that, Dox withdrawal was standardized for the cultures during following several passages (usually 2 weeks) unless other time points are indicated in the text. In this inducible system, we always test that insert expression is uniquely dependent on Dox and becomes absolutely undetectable after Dox withdrawal. As a control of the treatment itself, Dox was also added and tested in wild-type organoids.
Patient-derived organoids generation and culture
For this study, breast cancer patients (with BIRAD 4C-5-6) from Hospital 12 de Octubre (Madrid, Spain) donated one cylinder of the first core-needle tumor biopsy, prior diagnosis. To guarantee the protection of patients enrolled in this study, we have strictly followed the hospital guidance, the local regulations, the “Declaration of Helsinki” and the Guidelines of good clinical practice from the “International Conference on Harmonization” ICH E6 (R2), effective from June 14, 2017. The technical protocols for patient-derived sample collection and processing and any additional material delivered to the patient (such as Patient Information Sheets or the Informed Consent Document) were carefully evaluated and approved by the corresponding Clinical Research Ethics Committee, in accordance with national legislation. Tumor samples were immediately processed in our laboratory for organoid culture generation, as described above. We were able to maintain patient-derived organoid cultures for three or four passages, and the experiments were always performed at passage one. After 7 days of culture establishment and organoid amplification, patient-derived 3D cultures were transiently transfected with miR-203 mimics, followed by miR-203 withdrawal for three additional weeks. Hsa-miR-203 mimics were purchased from Sigma-Aldrich (MISSION microRNA mimics), and transient transfection was performed using Lipofectamine 2000 (Sigma), following manufacturer’s instructions. Since then, cultures were carefully evaluated under the bright-field microscope for quantification of organoid number and size, complexity, formation of 2D projections, and finally, immunofluorescence was performed at the end of the experiment (three weeks after the miR-203 brief exposure).
Immunofluorescence and immunohistochemistry
Organoids were fixed in 4% paraformaldehyde for at least 15 min, permeabilized using PBS 0.1% Triton X-100 for 15 min and blocked in BSA for 1 h at room temperature. Primary antibody incubation was performed overnight at 4ºC in all cases, followed by secondary antibody incubation for 1 h at room temperature. Nuclear staining was included in the last PBS wash, using Hoechst or DAPI. Primary antibodies used in this study were against CK8/18 (rabbit monoclonal EP17/EP30, Dako, IR094), CK14 (rabbit polyclonal AF64, Covance, PRB-155P) and E-cadherin (mouse monoclonal 36, BD Bioscience, 610182) for mouse-derived samples and CK8/18 (rat monoclonal, DSHB, 531826), CK14 (rabbit monoclonal, Abcam, ab181595) and vimentin (mouse monoclonal RV202, BD Pharmingen, 550513) for patient-derived samples. Cells were examined under a Leica SP5 microscope equipped with white light laser and hybrid detection.
For immunohistochemistry, tissue samples were fixed in 10% neutral buffered formalin (4% formaldehyde in solution), paraffin-embedded and cut at 3 µm, mounted in superfrost®plus slides and dried overnight. Consecutive sections were stained with hematoxylin and eosin (H&E) or subjected to immunohistochemistry using automated immunostaining platforms (Ventana Discovery XT, Roche or Autostainer Plus Link 48). Antigen retrieval was first performed with high or low pH buffer depending on the primary antibody (CC1m, Roche or low pH antigen retrieval buffer, Dako), endogenous peroxidase was blocked (peroxide hydrogen at 3%), and slides were incubated with primary antibodies against Ki67 (rabbit monoclonal D3B5, Cell Signalling Technology, 12202), cleaved Caspase 3 (rabbit, Cell Signalling Technology, 9661), CK5 (rabbit polyclonal AF 138, Covance, PRB-160P), SOX-10 (goat polyclonal N20, Santa Cruz Biotechnology, sc-17342), CD44 (rabbit polyclonal, Abcam, ab157107), H3K27me3 (rabbit monoclonal C36B11, Cell Signalling Technology, 9733), prolactin (rabbit polyclonal, Dako, A0569), progesterone receptor (rabbit monoclonal SP2, Thermo Scientific, RM-9102-R7), NeuN (mouse monoclonal A60, Millipore, MAB377), E-cadherin (mouse monoclonal 36, BD Bioscience, 610182), Aldh1/2 (mouse monoclonal H-8, Santa Cruz Biotechnology, sc-166362), CK8/18 (rabbit monoclonal EP17/EP30, Dako, IR094), CK14 (rabbit polyclonal AF64, Covance, PRB-155P), smooth muscle actin (mouse monoclonal 1A4, Dako, IR611), estrogen receptor alpha (rabbit polyclonal, Santa Cruz Biotechnology, sc-542).
Secondary antibodies were conjugated with horseradish peroxidase (OmniRabbit, Ventana, Roche), and the immunohistochemical reaction was developed using 3,30-diaminobenzidine tetrahydrochloride (DAB) as a chromogen (Chromomap DAB, Ventana, Roche or DAB solution, Dako) and nuclei were counterstained with Carazzi’s hematoxylin. Finally, the slides were dehydrated, cleared and mounted with a permanent mounting medium for microscopic evaluation. The images were acquired with a slide scanner (AxioScan Z1, Zeiss). Images were captured and quantified using the Zen Software (Zeiss).
Analysis of mRNA levels, RNA sequencing
RNA/microRNA was extracted from organoids samples with TRIzol (Invitrogen) or by using the miRVana isolation kit (Thermo Fisher), following the manufacturer’s recommendations and after the dissociation of Matrigel/BME from the cultures by using the Cell Recovery Solution (Corning), following the manufacturer’s protocols. For reverse transcription of microRNAs, we used the TaqMan small RNA assay (4366596), including the specific oligonucleotides for mmu-miR-203-5p and 3p (002580 and 000507), miR-16 and the housekeeping RNAs sno-202 or sno-142. Conditions for miRNA amplification were as follows: 30 min at 16ºC; 30 min at 42ºC and a final step of 5 min at 85ºC. Quantitative real-time PCR was then performed using the TaqMan Universal PCR Master Mix (434437) following the manufacturer’s instructions in an ABI PRISM 7700 Thermocycler (Applied Biosystems).
For RNAseq, total RNA was extracted using the miRVana miRNA isolation kit (Thermo Fisher), following the manufacturer’s recommendations. Between 0.8 and 1 µg of total RNA were extracted from organoids after dissociating the Matrigel/BME from the cultures (as indicated above). RIN (RNA integrity number) numbers were always in the range of 9 to 10 (Agilent 2100 Bioanalyzer). 250 ng of total RNA samples was used. Average sample RNA integrity number was 9.1 (range 8.2–9.8) when assayed on an Agilent 2100 Bioanalyzer. Sequencing libraries were prepared with the “QuantSeq 3’ mRNA-Seq Library Prep Kit (FWD) for Illumina” (Lexogen, Cat. No. 015) by following manufacturer instructions. This kit generates directional libraries stranded in the sense orientation, the read1 (the only read in single read format) has the sense orientation. Library generation is initiated by reverse transcription with oligo dT priming, and a second strand synthesis is performed from random primers by a DNA polymerase. Primers from both steps contain Illumina-compatible sequences. Libraries were completed by PCR, applied to an Illumina flow cell for cluster generation and sequenced on an Illumina HiSeq 2500 with v4 Chemistry by following manufacturer’s protocols. Read adapters and polyA tails were removed with bbduk.sh (
https://sourceforge.net/projects/bbmap/), following the Lexogen recommendations. Processed reads were analyzed with the nextpresso pipeline [
75], as follows: Sequencing quality was checked with FastQC v0.11.7 (
http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Reads were aligned to the mouse reference genome (GRCm38) with TopHat-2.0.10 [
76] using Bowtie 1.0.0 [
77] and Samtools 0.1.19 [
78] (library-type fr-secondstrand in TopHat), allowing two mismatches and twenty multihits. Read counts were obtained with HTSeq-count v0.6.1 [
79] (stranded = yes), using the mouse gene annotation from GENCODE (gencode.vM20.GRCm38.Ensembl95). Differential expression was performed with DESeq2 [
80], using a 0.05 FDR. GSEA Pre-ranked [
81] was used to perform gene set enrichment analysis for several gene signatures on a pre-ranked gene list, setting 1000 gene set permutations. Only those gene sets with significant enrichment levels (FDR q-value < 0.25) were considered.
Acknowledgements
We thank the CNIO Histopathology, Molecular Imaging and Bioinformatics Units, UCM Confocal Unit, CNIO and UCM Animal Facilities for their technical support. We are indebted to the members of the Cannabinoid Signaling group (UCM) and the Cell Division and Cancer group (CNIO) for their constant support and advice. We are grateful to the Hospital 12 de Octubre, particularly to the Radiology, Gynecology and Oncology Units for their constructive participation in this work and to the patients enrolled in this research study, who kindly donated one cylinder of their core-needle biopsy for the development of this project. This work has been in part financed by benefactors, through the crowdfunding project “Match point against breast cancer” (PRECIPITA PR242, 2019; FECYT; Spanish Ministry of Science and Innovation, MICINN, led by MS-R), and donations to Asociación Española contra el Cáncer (AECC). We are extremely thankful to all our donors. The work has been also funded by the Spanish Ministry of Science and Innovation and the Ministry of Economy and Competitiveness (supported with European Regional Development funds): PID2021-128726 to MM, CNS2022-135364 to MS-R, PI20/00590 to CS, as well as by Comunidad de Madrid (Y2020/BIO-6519 and S2022/BMD-7437) to MM. MS-R was supported by AECC (AIOA120833SALA and INVES18005SALA), a Juan de la Cierva incorporación and a Ramón y Cajal contract (RYC2020-028929-I, from the MICINN, FSE/ Agencia Estatal de Investigación). NGM-I was supported by AECC (PRDMA19003GARC).
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