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Patient-derived normal and tumor colorectal organoids: Studies on gene expression, vitamin D and drug activity

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Deaprtamento Bioquímica Facultad de Medicina Universidad Autónoma de Madrid

Patient-derived normal and tumor colorectal organoids:

Studies on gene expression, vitamin D and drug activity

Alba Costales Carrera

Licenciada en Biología, Msc. en Bioingeniería

Directores de Tesis:

Alberto Muñoz Terol Antonio Barbáchano Becerril

Instituto de Investigaciones Biomédicas “Alberto Sols” Madrid

Consejo Superior de Investigaciones Científicas-Universidad Autónoma de Madrid.

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Prof. Alberto Muñoz Terol, Profesor de Investigación del C.S.I.C en el Instituto de Investigaciones Biomédicas “Alberto Sols” y el Dr. Antonio Barbáchano Becerril

CERTIFICAN: que Alba Costales Carrera, licenciada en Biología por la Universidad de Oviedo con un Máster en Bioingeniería por el Institut Químic de Sarriá, ha realizado bajo su dirección el trabajo titulado:

Patient-derived normal and tumor colorectal organoids:

Studies on gene expression, vitamin D and drug activity

Y que el trabajo cumple todas las condiciones requeridas por la legislación vigente de originalidad y calidad científica para ser presentado y defendido con el fin de optar al grado de Doctora.

Para que conste donde proceda, firmamos el presente certificado.

En Madrid, a 25 de febrero de 2019,

Alberto Muñoz Terol Antonio Barbáchano Becerril

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ACKNOWLEDGEMENTS

Esta Tesis se ha realizado gracias a un Contrato de Formación del Personal Investigador del Ministerio de Economía, Industria y Competitividad Asociado al proyecto SAF2013-43468-R, SAF2015-71878-REDT/Nurcamein, SAF2016-76377-R así como de una Ayuda para la realización de Estancias breves en el Extranjero disfrutada en el grupo del Prof. Carsten Carlsberg de la Eastern Finland University de Kuopio, Finlandia.

Agradecemos a la Dra. Burgos por ser gran endoscopista, persona y siempre tener tiempo y una sonrisa para nosotros (incluso tras 30 horas de guardia). Al personal Biobanco del Hospital Universitario La Paz, que se crecen ante la falta de recursos y hacen un trabajo espectacular. A la Prof. M.Demay por cedernos los ratones Vdr-, a G.Gónzalez-Bueno por su ayuda con el mantenimiento/genotipado de los ratones y su contacto para el análisis mutacional de algunos cultivos de organoides (con trampa y error detectados, así de gusto navegar en ciencia). A H.G.Pálmer por el análisis mutacional de algunas otras muestras, a M.T.Berciano por su ayuda con los estudios de microscopía electronica. Al Prof. M.Lafarga por sus magistrales clases telefónicas sobre ultraestructura cellular y por enseñarnos a reconocer “Cuerpos de Cajal”, es un un privilegio escucharle. Al Dr.Luis del Peso por su ayuda bioinformática y por esforzarse en entender a fondo cada proyecto e intentar sacar siempre lo mejor a pesar de que entre todos le volvamos loco. Al Msc.Orlando Domínguez por extinguir nuestras alarmas con calmadas explicaciones razonadas sobre secuenciación.

“A hombros de gigantes” me siento yo, a nivel científico y personal. Imposible agradecer a todas las personas que me han apoyado para estar hoy escribiendo estas líneas.

Empezando por lo más cercano, gracias a Asun y a Toño por haberme enseñado el arte de los organoides de manera tan exquisita, tan dedicada, con esa paciencia, impertérrito buen humor y siempre buenas palabras (incluso aquella vez que asesiné por olvidarme de echar medio no una, sino ¡dos veces! aquel cultivo “de tumor 68” tan importante). Ha sido un placer compartir con vosotros cada día, Greta va a ser muy afortunada de teneros, crecerá superfeliz y hará el mundo un lugar mejor. A nuestra querida Pilar, por traer brisa fresca, por tu eterna sonrisa y alegría que transmites allá donde vas, ¡por eso te salen todos los experiementos!.

A Gemma y a Chus por siempre estar ahí para echar una mano, he disfrutado mucho también de vuestra compañía, charlas, luchas por el medio ambiente y por los animales, penas compartidas (que siempre son menos severas) sobre atascos, vinos/tapas en ”Pepe el Guarro” (y otros…) y por todos los momentos del día a día.

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¡y además venir a verme de mochileros y hacerme disfrutar!. Por confiar en mí y apoyarme en todo, siempre, incondicional. Soy la hija mas afortunada del mundo. A Veli y Jose por haberme adoptado y haberme tratado siempre como una hija más, estáis en muchos de mis mejores recuerdos, me siento tan fortunada y orgullosa de tener una famila así… A Esther y Diego por acogerme cuando era una “sin techo” y cuando soy una “con techo” también; por la confianza, la generosidad, la alegría y por hacerme sentir en casa a 500 km de Gjón. A Claudia, Elsa y Vega por hacerme reír tanto viendo el mundo como una niña otra vez y animarme a defender la tesis cual Kung-Fu panda (lo haré). A Pablo, Bea Valle y Vic, porque sois geniales y tengo ganas de veros más. A mi tía Isa y tío Robin, por ser tan reflexivos, innovadores, quererme y enseñarme tanto desde el “Folindón” hasta hoy, con vosotros siempre genial. A Eladio y Alicia por ser tan auténticos, divertidos, cariñosos y gente excepcional. A mi abuelo Eladio por todo y a mi abuela Avelina, pionera en perseguir sus sueños aunque tuviese que entrar por la Puerta de servicio para estudiar por ser mujer de otra época. Gracias también a Oles (cuna de mis mejores momentos) y cuna de la spectacular familia que tengo.

Por ultimo y más importante (en ciencia al menos) gracias a Alberto, por su dedicación, su cercanía, su paciencia, su pesimismo (que obliga al optimismo, así que es genial) y por su integridad. Espero esta tesis no acabe con tu buen character, habrá que compensarlo con un vino.

Y como no, a Otín, inspiración y aspiración de cualquiera, gracias por hacer de guía y Celestino.

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SUMMARY

Colon/colorectal cancer (CRC) is the most frequent neoplasia in Spain (both genders), a leading cause of cancer-related deaths worldwide and the cancer most associated to vitamin D deficiency in epidemiological studies. Previous mechanistic studies have shown a variety of effects of the active vitamin D metabolite, 1alpha,25-dihydroxyvitamin D3 (calcitriol), on human colon carcinoma cells and, recently, on stromal fibroblasts. This Thesis constitutes the first study on the action of calcitriol on human colon and rectum stem cells, both normal and tumoral.

Results obtained show that the high affinity vitamin D receptor (VDR) is expressed by a large proportion of human colon stem cells (LGR5+) located at the crypt bottom in healthy tissue, as well as by organoids generated by these cells and by those derived from colon tumors. In both types of organoids, calcitriol inhibits cell proliferation without affecting clonogenicity.

In normal colon organoids, RNA-sequencing analysis have led to the identification of a high number of genes regulated by calcitriol including some involved in stemness (LGR5, MSI1, SMOC2,...), proliferation control (LRIG1...) and other cell functions. Chromatin immunoprecipitation-sequencing studies have led to the identification of genes that contain VDR binding sites in or close to their promoters and so, are candidate direct transcriptional calcitriol targets. In tumor organoids, calcitriol also regulates a wide array of genes, which partially overlap with those in normal organoids. Contrarily to normal organoids, calcitriol targets in colon tumor organoids include differentiation genes but only few genes related to stemness. In line with these findings, ultrastructural analyses by electron microscopy show that calcitriol does not alter the undifferentiated cell phenotype in normal colon organoids but, instead, it induces differentiation features in tumor organoid cells. Altogether, this pioneering study demonstrates that calcitriol distinctly regulates the phenotype of human colon normal and cancer stem cells. Our results show also that human (left-side) colon and rectum normal organoids have very similar patterns of gene expression, and that, accordingly, calcitriol has comparable effects in the types of organoids.

Finally, we have set up an assay for antitumor drugs using patient-derived colon tumor organoids that may be useful for personalized medicine of CRC patients and for the analysis of the activity of new compounds.

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RESUMEN

El cáncer de colon/colorrectal (CCR) es la neoplasia más frecuente en España (ambos sexos), una de las principales causas de muertes relacionadas con el cáncer en todo el mundo y el cáncer más asociado a la deficiencia de vitamina D en los estudios epidemiológicos. Estudios previos han revelado efectos protectores del metabolito activo de la vitamina D, la 1a,25-dihidroxivitamina D3 (calcitriol) en células de carcinoma de colon humano y, recientemente, en fibroblastos estromales. Los organoides son estructuras 3D in vitro derivadas de células troncales/stem que reproducen parcialmente la organización tisular y permiten el estudio de procesos biológicos. Esta Tesis constituye el primer estudio sobre la acción del calcitriol en organoides normales y tumorales derivados de muestras de pacientes con CRC.

Los resultados muestran que el receptor de vitamina D (VDR) se expresa tanto en las células troncales del tejido colónico humano sano como en organoides generados a partir de estas células, y también en organoides derivados de algunos tumores de colon. En ambos organoides, normales y tumorales, el calcitriol inhibe la proliferación celular sin afectar la clonogenicidad. En los organoides normales, análisis transcriptómicos globales (RNA-seq) han identificado genes regulados por calcitriol, algunos implicados en el aumento de troncalidad/stemness, control de la proliferación y otras funciones. Los estudios de inmunoprecipitación de cromatina acoplada a secuenciación (ChIP-seq) han permitido identificar genes con sitios de unión de VDR estimulados por calcitriol, probables dianas directas de la vitamina D. En organoides tumorales el calcitriol regula también un amplio conjunto de genes, algunos coincidentes con los identificados en organoides normales (escasos relacionados con stemness) y otros específicos, entre ellos genes de diferenciación. En línea con estos datos, análisis mediante microscopía electrónica muestran que el calcitriol no altera el fenotipo de células indiferenciadas en los organoides normales de colon, y, por el contrario, induce diferenciación celular en organoides tumorales. Este estudio pionero demuestra que el calcitriol regula de modo diferencial el fenotipo de las células troncales/stem normales y tumorales de colon humano.

Estudios comparativos han mostrado que los organoides normales derivados de tejido sano de colon y recto tienen patrones de expresión génica casi idénticos. De acuerdo con ello, el calcitriol tiene efectos similares en ambos tipos de organoides. Organoides tumorales derivados de colon y recto tienen perfiles transcriptómicos más heterogéneos, si bien su respuesta a calcitriol es muy similar.

Finalmente, hemos puesto a punto un ensayo de drogas antitumorales utilizando organoides tumorales de colon derivados de pacientes con CCR, que ha permitido confirmar la utilidad de este sistema en medicina de precisión/personalizada y para el estudio de nuevas moléculas.

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INDEX

INTRODUCTION... 1

Colorectal cancer ... 1

CRC genetics ... 1

The Cancer Stem Cell model ... 2

Wnt/β-catenin signaling pathway and its relevance in CRC ... 3

The intestinal epithelium ... 4

Normal and Cancer Stem cells ... 5

Stem cells, subpopulations and markers ... 6

Other stem cell markers in human colon crypts ... 7

Colon cancer and rectal cancer, two different entities ... 8

Vitamin D and colorectal cancer ... 9

Effect of calcitriol on human colonic fibroblasts ... 14

Organoids ... 14

Tumor organoids ... 18

Organoids as an antitumor drug assay platform ... 19

Towards precision medicine ... 20

OBJECTIVES ... 23

OBJETIVOS ... 25

MATERIALS AND METHODS ... 27

Human samples ... 29

Study approval ... 29

Cell culture and Living Biobank ... 29

Cell lines ... 29

Living Biobank ... 29

3D cultures of normal and tumoral colon organoids derived from Surgical Resection Samples ... 30

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Establishment 3D cultures of normal colon organoids derived from Vdr-/- and Vdr+/+

mice ... 33

Growth and expansion of organoid cultures... 33

Wnt3a-conditioned medium ... 34

Proliferation and clonogenicity assays ... 34

Drug-assays ... 35

Characterization assay for patient-derived organoids ... 35

Organoid Drug-assay ... 37

Drug-assay used only for calcitriol-drug interaction tests. ... 38

RNA techniques ... 39

Gene Expression Microarray analysis ... 39

Real-time quantitative PCR (RT-qPCR) ... 39

RNA-sequencing (RNA-seq) ... 40

VDR interference ... 40

RNAscope in situ hybridization ... 41

DNA techniques ... 42

Mutational status ... 42

Chromatin immunoprecipitation-sequencing VDR (ChIP-seq) ... 42

Protein Techniques ... 43

Western blot ... 43

Immunohistochemistry ... 44

Electron microscopy ... 44

Bioinformatic processing and statistics ... 45

Functional enrichment analysis... 45

Data availability ... 45

Mouse microarrays ... 45

RNA-seq ... 45

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Statistics ... 46

RESULTS ... 47

3.1. Chapter 1. Human colon normal and tumor organoids. Calcitriol effect ... 49

Graphical abstract Chapter 1. ... 49

3.1.1. Building a Living Biobank of human colon organoids from surgical samples.... 50

3.1.2. Normal and tumor colon stem cells express VDR and respond to calcitriol ... 54

3.1.3. Calcitriol Gene regulatory action in colon organoids: transcriptomic studies .. 59

3.1.4. Identification of direct calcitriol-target genes in human normal colon organoids ... 67

3.1.5. GSEA analysis supports a regulatory action of calcitriol on cell stemness and proliferation ... 73

3.1.6. Calcitriol differentially modulates cell phenotype and proliferation in normal and tumor colon organoids ... 74

3.2 Chapter II. Human normal rectum and rectal tumor organoids. Calcitriol effect .... 79

Graphical abstract. Chapter II... 79

3.2.1. Building a Living Biobank of human organoids from endoscopy samples ... 80

3.2.2. Normal colon vs normal rectum organoid gene expression analysis... 81

3.2.3. Normal rectum vs rectal tumor organoid gene expression analysis ... 82

3.2.4 Rectal tumor vs colon tumor organoid gene expression analysis ... 82

3.2.5. Effect of calcitriol on normal colon, normal rectum and rectal tumor organoids obtained from endoscopy samples. ... 87

3.3. Chapter III. Human colon organoids as an assay platform for antitumor drugs ... 95

3.3.1. Optimization of drug-assay parameters for each patient ... 95

3.3.2. Drug assay design ... 96

3.3.3. Z-score as quality control ... 97

3.3.4. Proof-of-concept using antitumor drugs in the clinical setting ... 97

3.3.5. Drug - calcitriol interactions... 101

DISCUSSION ... 103

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Chapter1. Calcitriol regulates human colon stem cells ... 106

Chapter 2. Comparative analysis of Colon/Rectum and Normal/Tumor organoids .. 111

Normal colon vs. normal rectum organoids ... 111

Colon tumor vs. rectal tumor organoids ... 113

Calcitriol effects in organoids ... 114

Chapter 3. Drug assays using patient-derived organoids ... 115

Limitations and future perspectives of organoid technology ... 116

CONCLUSIONES ... 119

CONCLUSIONS ... 12121

REFERENCES ... 12123

SUPPLEMENTAL TABLES ……….135

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LIST OF ACRONYMS / ABBREVIATIONS

5-FU. 5-fluorouracil

ASCL2. Achaete-scute homolog 2 BMI1. Polycomb complex protein BMI-1 BMP. Bone Morphogenic Protein

CA2. Calmodulin-dependent protein kinase II CAF. Cancer-Associated Fibroblasts

ChIP-seq. Chromatin Immunoprecipitation coupled to sequencing CMS1/2/3/4. Colorectal Molecular Subtypes 1, 2, 3 or 4.

CPM. Counts per million CRC. Colorectal cancer CSC. Cancer Stem Cell

DCSC. Deep-crypt secretory cells DE. Differentially expressed DKK. Dickkopf

DTT. Dithiothreitol

EDTA. Ethylenediaminetetraacetic acid EGF. Epithelial growth factor

FBS. Fetal bovine serum

HOPX. Homeodomain-only protein

LGR5. Leucine-rich repeat-containing G-protein coupled receptor 5 LRC. Label-retaining cells

LRIG1. Leucine-rich repeats and immunoglobulin-like domains protein 1 LRP. Lipoprotein receptor-related protein

NAF. Normal Associated Fibroblasts PDTX. Patient Derived Tumor Xenograft PDX. Patient Derived Xenograft

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RNF43. Ring finger protein 43 RSPO. R-spondin

RT. Room temperature

SFRP. Secreted frizzled-related protein

SMOC2. SPARC-related modular calcium-binding protein 2 TA. Transient Amplifying

VDR. Vitamin D Receptor ZNRF3. zinc and ring finger 3

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INTRODUCTION

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Colorectal cancer

Colorectal cancer (CRC) is the second (in women) and the third (in men) most common cancer type worldwide, and the neoplasia with highest incidence (both genders) in Spain, with a global 5-year survival rate of 64%. The incessant increase in CRC incidence rate in the last three decades in developed countries points to diet as an important risk factor, since intestinal tissue is directly exposed to toxins, xenobiotic and diet-conditioned variable intestinal microbiota, infections and inflammation.

Most CRCs are sporadic due to accumulation of mutations throughout life in proto- oncogenes and tumor suppressor genes (CRC Suppressor pathway), and usually appear above 50 years of age, providing a margin for prevention campaigns and early diagnosis. In addition, there are hereditary CRC syndromes (5-10%) characterized by early-age appearance [1]. The main two are familial adenomatous polyposis, caused by the inheritance of a mutated allele of the tumor suppressor gene Adenomatous Polyposis coli (APC), and hereditary non-polyposis colorectal cancer or Lynch syndrome, caused by the mutation or epigenetic silencing of genes involved in DNA mismatch repair such as MSH2, MLH1 and, less frequently, MSH6/GTBP, PMS1 and PMS2, which leads to microsatellite instability (CRC Mutator pathway).

CRC genetics

CRC is the solid tumor whose genetics is best known. Mutations leading to sporadic CRC affect genes of stemness and pathways of proliferation/differentiation control. The mutation of APC or, less frequently and in a mutually exclusive manner, CTNNB1/β-catenin, AXIN, RSPO2/3 or TCF7L2/TCF4 causes the aberrant activation of the Wnt/β-catenin signaling pathway leading to polyp/adenoma formation. This is followed by the accumulation of other mutations that cause progression to carcinomas (Figure 1). Thus, a high percentage of colon adenomas have mutations in KRAS or BRAF and inactivating mutations of genes of the transforming growth factor (TGF-β) pathway such as SMAD4, SMAD2 or TGFβR2 that confer additional malignant features to adenomas. Inactivation of TP53 is associated with the adenoma-carcinoma transition in 50% of CRC, and a high percentage of CRC loses the expression of ephrin B receptors (EPHB) during this transition. Of note, the deletion of the c-Myc gene in Apcmin mice (a model of intestinal tumorigenesis with a mutated germline Apc allele) blocks the progression from adenoma to carcinoma, indicating a critical role of c-Myc, a Wnt/b-catenin target gene, in the initial stages of this neoplasia. In The Cancer Genome Atlas consortium study (2012), the analysis of 224 cases with CRC distinguished between non-hypermutated CRC (84%) with <8.24 mutations per 106 bases and hypermutated CRC (16%) with >12 mutations per 106 bases. Non-hypermutated CRC

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followed the Suppressor pathway (Figure 1) and are characterized by chromosomal instability and microsatellite stability. Among hypermutated CRC, 75% were enriched for microsatellite instability and CpG island methylator phenotype, whereas the remaining 25% presented somatic mutations in mismatch repair or polymerase e (POLE) genes, showing >40 mutations per 106 bases. The Wnt/β-catenin signaling pathway was constitutively activated in 93% of non- hypermutated CRCs and 97% of hypermutated CRCs [2]

Figure 1. CRC pathways. Genetic alterations associated with sporadic tumor progression and its frequency.

Modified from [3].

Recently, non-stepwise models of CRC progression and intratumoral heterogeneity have been proposed based on high level genomic/chromosomal damage acquired in single or few drastic events such as Big-Bang models (chromothripsis, chromoplexy, mitotic catastrophy) and the Cancer Punctuated Equilibrium model [4].

The Cancer Stem Cell model

The high level of cell proliferation needed for the extensive renewal of the colonic epithelium is probably the origin of the high frequency of mutations and, therefore, the appearance of CRC.

In this scenario, stem cells and their proliferative early progeny are candidates to be the origin of this neoplasia. According to this theory/model, stem cells altered by genetic changes (mutations) and epigenetic alterations (Cancer Stem Cells, CSC) are the initiators of the tumorigenic process [5,6]. Cancer driver gene mutations allow the mutated stem cells to thrive independently of their native niche constraint being proposed as key in tumor progression, the formation of metastasis, and resistance/recurrence to antitumor therapy [7]. Thus, based on numerous studies in experimental animals, it has been proposed that the alteration of stem cells is a main factor in human CRC [8].

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Wnt/β-catenin signaling pathway and its relevance in CRC

In normal epithelial cells (Figure 2A), the absence of Wnt factors leads β-catenin protein to bind to E-cadherin at the adherens junctions, whereas free cytosolic b-catenin binds to APC and AXIN ending up degraded by the proteasome.

The stimulation by Wnt (Figure 2B) prevents the formation of the degradation complex of β- catenin, causing its accumulation in the cytoplasm and its partial translocation to the cell nuclei.

In the nuclei, β-catenin regulates the expression of hundreds of genes involved in proliferation, migration and stemness. In intestinal stem cells this pathway has additional actors: R-spondins are a 4-member family of proteins that potentiate the Wnt pathway as they bind to plasma membrane LGR4/5 stabilizing the Wnt receptors LRP and Frizzled via the inhibition of RNF43 and ZNRF3 ubiquitin ligases [9–11].

Mutations of APC, CTNNB1/β-catenin or AXIN are present in most CRCs (Figure 1) preventing the formation of the β-catenin degradation complex and causing the constitutive activation (independent of Wnt factors) of the pathway, which favours an undifferentiated proliferative phenotype and promote tumor progression (Figure 2B). In >80% of CRCs the constitutive activation of the Wnt pathway is due to mutations in APC, less frequently, and only in tumors that do not have mutated APC, mutations are observed in CTNNB1/β-catenin or in AXIN, RSPO2/3 or RNF43 (Figure 1). In addition to these mutations, local overexpression of Wnt factors and/or silencing of genes that encode Wnt pathway inhibitors (SFRPs…) also contribute to abnormal cell survival and proliferation.

The analysis of mice with an altered Wnt/β-catenin pathway (Apcmin or mice expressing a constitutively active form of β-catenin) confirms the activation of this route as main driver of intestinal tumorigenesis.

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Figure 2. Wnt/β-catenin or canonical Wnt pathway in stem cells. A) In the absence of Wnt factors, β- catenin is associated with a cytoplasmic complex containing the proteins APC, Axin and the CKI and GSK3β kinases.

These phosphorylate β-catenin and mark it for ubiquitination and subsequent degradation by the proteasome. B) Wnt potentiated by R-spondin factors, inhibit the phosphorylation and ubiquitination of β-catenin, which accumulates in the cytoplasm and translocates to the nuclei. There, it is associated with proteins of the TCF/LEF family (T-cell factors/Lymphoid enhancer factor) and activates the transcription of proliferation, migration and stemness genes. Modified from [12].

The intestinal epithelium

Mammalian intestine is internally covered by a mucosal layer built of epithelial cells responsible for the absorption of water and nutrients and for the isolation of the organism from toxic substances and pathogens. Colon and rectum are the last sections of the intestine whose main function is the absorption of water, ions and some vitamins. The mucosa of colon and rectum (Figure 3) is composed of an invaginated epithelium shaping tubular glands open to the digestive tract lumen, named crypts of Lieberkühn. Unlike the small intestine, colon and rectum lack microvilli and Paneth cells in their crypts.

Intestinal epithelium renewal rate is very high (4-5 days) and results from the proliferation of stem cells (located in the bottom of the crypts) and their progeny called transient amplifying cells (TA), located in the medial part of the crypt (Figure 3B). As cells divide they are pushed towards the upper zone of the crypt were they fully differentiate and are swept along with digestion debris.

Differentiation goes towards goblet/mucosecretory cells, that secrete mucus to lubricate and facilitate the transit of feces; enterocytes/absorptive cells, which secrete hydrolases and absorb water and nutrients; and a small number of enteroendocrine cells (deep crypt secretory cells, DCSC) that secrete molecules with antimicrobial and hormonal action.

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Figure 3. General outline of the colon. A) Scheme of the colonic epithelium. The epithelial surface invaginates into the submucosa forming the crypts. (B) Immunohistochemistry of a colonic crypt. Stem cells are located in the crypt bottom, proliferating TA cells in the medial zone, and the differentiated cells in the upper part facing colon lumen. Proliferative cells are stained with Ki67 marker (brown nuclei). Modified from [13] and Encyclopædia Britannica.

Normal and Cancer Stem cells

Pluripotent undifferentiated cells located in the bottom of the crypts have the capacity to produce all the intestinal epithelium cell types, as well as the ability for self-renewal. Their pluripotency was demonstrated by lineage tracing studies (Figure 4-A, B, C) where from a single cell from a special Lgr5+ cell population (green in Figure 4 D and E), a complete functional crypt was renewed including all mouse tissue cell types.

Figure 4. Lgr5+ cells in mouse colonic crypts. A, B and C) Lineage tracing studies in mice.

A single Lgr5+ stem cell (LacZ) proliferates to produce a complete crypt containing all characteristic cell types. D, E) Location of GFP labeled Lgr5+ stem cells in mice crypts [14]

Niche signal gradients determine the fate of stem cells and their progeny. EGF is the main proliferation agent; and Wnt, R-spondins, Notch, Hedgehog, Hippo and BMP (bone morphogenetic proteins) are the main signaling factors that govern the cellular order in the intestinal crypts. Their expression gradients (relative to the organization of the colonic crypt nearby) are sketched in Figure 5 (middle).

A B

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Wnt and R-Spondins are essential for the maintenance and proliferation of stem cells.

Both are produced by stromal cells (pericryptal/subepithelial myofibroblasts and perhaps other mesenchymal cells) and probably by DCSC (Figure 5) near the base of the crypt (in the small intestine by Paneth cells). Hedgehog is more intensively expressed in the crypt apical zone and antagonizes Wnt signaling. BMPs, mainly BMP-4, induce cell differentiation [15] and are secreted by differentiated apical cells producing a gradient like that of Hedgehog (Figure 5). In the crypt basal zone, the presence of Noggin (an extracellular inhibitor of BMP) favors the action of Wnt factors. Notch signaling is greater at the crypt base and is required to maintain stemness. Notch inhibition (in the absence of Wnt) produces apical zone differentiation towards the secretory lineage [7].

Figure 5. Diagram of colonic crypts, cell types and gradients of factors that influence stemness / differentiation. In the basal zone of the crypt are the Lgr5+ stem cells, within which there are more quiescent

"reserve" subpopulations (such as +4 or Mex3a high). Wnt factors are concentrated in the basal zone of the crypt promoting stemness. Notch regulates cell proliferation and differentiation and its concentration gradient is higher in the basal zone. BMP promotes differentiation and concentrates in the apical zone of the crypts. Modified from [16]

Stem cells, subpopulations and markers

The lack of functional stem cell markers and assays for demonstrating stemness stagnated the stem cell field for years, until Hans Clevers’ group identified and demonstrated through lineage tracing studies that Lgr5-expressing cells (Lgr5+) were pluripotent stem cells [14,17]. Since then the research field is boiling. Different populations of stem cells with distinctive proliferation rates have been characterized (Figure 5-right). In mice, high expression of Mex3a defines a subpopulation of Lgr5+ cells able to give rise to all cell types but with reduced proliferative capacity [18], and are considered the "reserve" stem cells (Figure 5, red). Another subpopulation of Lgr5+ cells are the "+4 label retaining cells" (+ 4), named for their position with respect to the crypt bottom (Figure 5, blue) and their high quiescence[19]. The +4 cells migrate to the crypt base becoming

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Lgr5+ proliferating cells that regenerate the entire crypt in case of ablation of the latter or tissue damage [20,21].

Growing evidence supports the idea of stemness being a cellular status instead of a defined cell type. Several studies show that all types of differentiated cells from the intestinal crypt are able to dedifferentiate and transdifferentiate to recover stemness and regenerative competency, putatively in response to stromal signals [22–24]. This unexpected very high phenotypic plasticity is considered a driver of cancer initiation, progression and therapy resistance [25], pointing against therapies targeting LGR5 alone and, instead, suggesting the necessity of the combination of therapies anti-LGR5 and anti-stromal/niche factors that induce plasticity, in situ cancer stem cell clonogenicity and tumor expansion in CRC [22].

Other stem cell markers in human colon crypts

Intestinal stem cell markers additional to LGR5 have been characterized or proposed (Table 1), some of them have been confirmed by lineage tracing studies. Among them:

- SMOC2 (secreted modular calcium-binding protein-2), analogously regulated to LGR5 and expressed in the basal zone of the Lieberkühn crypts.

- LRIG1, a quiescent stem cell marker that is a pan-ERBB/tyrosine kinase negative inhibitor and tumor suppressor.

Table 1. Proposed intestinal stem cell markers. Some genes have been confirmed by lineage tracing studies (Y = yes; N = no; NR = non-reported). Modified from [26]

Active-cycling cells Study Lineage tracing confirmation

Lgr5 (Barker et al. 2007)[14] Y

Ascl2 (Van der Flier et al. 2009)[27] NR

Olfm4 (Van der Flier et al. 2009)[28] NR

Lrig1 (Wong et al. 2012)[29] Y

Sox9lo (Formeister et al. 2009)[30] NR

CD24lo (von Furstenburg et al. 2011)[31] NR

Upper SP (von Furstenburg et al. 2014)[32] NR

CD44+ CD24lo CD166+ GRP78lo (Wang et al. 2013)[33] NR

Smoc2 (Munoz et al. 2012)[11] Y

Troy (Fafilek et al. 2013)[34] NR

Msi1 (Itzkovitz et al. 2011)[35][36] NR

Ptk7 (Jung et al. 2015)[37] N

Slow-cycling/quiescent cells

Bmi1 (Sangiorgi & Capecchi, 2008)[38] Y

mTert (Montgomery et al. 2011)[39] NR

Hopx (Takeda et al. 2011)[40] Y

Lrig1 (Powell et al. 2012)[41] Y

Dclk1 (May et al. 2008)[42] NR

Sox9hi (Roche et al. 2015)[43] NR

Lower SP (von Furstenburg et al. 2014)[32] NR

LRC (Buczacki et al. 2013)[32] NR

Wip1 (Demidov et al. 2007)[44] NR

Krt19 (Asfaha et al. 2015)[45] NR

Mex3ahi (Batlle et al. 2017)[18] NR

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8

Colon cancer and rectal cancer, two different entities

Colon and rectum are part of the same organ but have different embryological, anatomical and physiological characteristics. Advances in oncological medicine and the accumulation of clinical data are increasingly shaping the treatment of CRC. Colon and rectal tumors are often referred to as "colorectal cancer/CRC"; however, tumors of these two regions are managed differently in the clinic.

Usually, rectal tumors are treated with radiotherapy and chemotherapy prior to surgical resection of the tumor in order to reduce its size and so facilitate surgery. At 6 - 8 weeks after surgery, chemotherapy is used again to reduce the risk of recurrence, usually a FOLFOX regimen (5-FU, oxaliplatin and leucovorin), FOLFIRI (folic acid, 5-FU and irinotecan) or other combinations depending on the specific characteristics of the patient. Treatment of rectal tumors evolves towards eliminating surgical resection and using only chemotherapy, as survival rates at 4 years in patients who underwent surgical resection are 95%, not far from 91% survival for patients who have only been treated with chemotherapy [46,47].

In contrast, surgical resection is the first and main step in the treatment route of colon tumors At 6 - 8 weeks after the intervention, patients are treated with chemotherapy in FOLFOX, FOLFRI or other drug combinations that contain irinotecan or oxaliplatin.

Remarkably, several studies have demonstrated that the mutations in the two locations, colon and rectum (analyzed in global samples, not in isolated or specific cell populations, and even in different segments of the colon), differ, and, moreover, their respective risk factors are also distinct [48–54].

In line with this, CRCs have been recently classified in four molecular subgroups with distinct mutations and phenotypic characteristics whose frequencies vary along the colon [55] (Figure 6).

Figure 6. Molecular classification of CRCs. CRCs have recently been classified into 4 types (CMS1,2,3 and 4) according to their mutational and phenotypic characteristics. The frequency of CRC classes varies in the different portions of the colon, with right colon tumors being very different from those in the left or rectum colon.

Modified from.[55]

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Vitamin D and colorectal cancer

Vitamin D has attracted great attention in oncology given the large number of epidemiological studies suggesting the link of its deficiency with high risk of developing multiple cancers [56,57]. In addition, experimental studies in cultured cells and animals have revealed effects that are compatible with an anticancer activity in vivo. CRC is the neoplasia mostly associated to this potential beneficial action of vitamin D in terms of incidence and/or mortality and, furthermore, vitamin D has low toxicity and low cost as compared to current therapies [57–

60]

In 1941, F. Apperly, an American physician and pathologist, demonstrated an inverse correlation between the level of ultraviolet solar irradiation and cancer mortality rate, and proposed that sunlight in some way conferred "relative immunity to cancer" (in organs other than skin) [61]. His article did not attract much attention until in 1980 when C. Garland and F. Garland [62]

proposed vitamin D as responsible for that correlation. Since then, “Vitamin D” related publications have continued rising, and up to more of 4,600 papers on this topic were published last year (Figure 7).

Up to 90% of vitamin D in our organism is synthesized in the skin by action of solar UV radiation (280-315 nm) causing a photochemical reaction that rearrange the 7-dehydrocholesterol bonds producing cholecalciferol (Figure 8). This molecule has no biological activity, and it needs two consecutive hydroxylations, first in the liver to form 25-hydroxyvitamin D3 or calcidiol and then in the kidney, many epithelial cells and immune cell types, to achieve its active form, 1alpha,25- dihydroxyvitamin D3 or calcitriol.

Figure 7. Articles published (1996-2018) about Vitamin D appearing in PubMed.

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10

Figure 8. Vitamin D system. UV radiation of 280-315 nm wavelength produces a photochemical reaction in the skin that converts the precursor into vitamin D (inactive form). Two consecutive hydroxylations in residues 1 and 25 are necessary to reach the active form of vitamin D, named calcitriol.

In a latitude-dependent fashion, the intensity of the solar radiation is not enough all-year- round to synthesize adequate vitamin D levels in the skin. Thus, a large part of the population is vitamin-deficient during part of the year, a situation that could/should be prevented by incorporating (“fortifying”) the diet with vitamin D or by taking vitamin D supplements. Vitamin D status is analyzed by measuring blood levels of calcidiol, (nM range) that is much more stable (half-life of around three weeks) than calcitriol (pM range, half-life of around three hours). There is no consensus about minimal or optimal calcidiol concentrations. Usually, <10 ng/ml (25 nM) is considered severe deficiency, >10 to <20 ng/ml as deficiency, 20 to 30 ng/ml as insufficiency (or normal situation for some authors) and >30 ng/ml sufficiency. Toxicity can be considered at levels superior to those naturally occurring in populations living at Equator latitude (around 50 ng/ml) that receive extensive solar irradiation.

Human interventional studies

A large body of literature supports a protective role of vitamin D against cancer. Serum 25(OH)D levels >40 ng/ml were associated with 65% lower risk of all invasive cancers [63]. The strongest evidence in epidemiological studies is for solar radiation and levels of calcidiol/25(OH)D in serum inversely correlated with CRC risk. Calcidiol concentrations >32 ng/ml in blood are associated with a 50% reduction in CRC risk [64]. A recent meta-analysis (2017) using data from 2,900 CRC patients show a better recurrence-free prognosis and a better overall survival of patients with higher levels of calcitriol.

However, there is a strong debate about whether vitamin D status is linked with disease or is just a correlate marker of overall health. In line with population studies analysing circulating calcidiol concentrations, evidence from in vitro and animal model studies supports an

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antineoplastic role of vitamin D. In contrast, randomized controlled trials have yielded mixed, inconsistent results, which have been proposed to derived from their bad design (low doses, short duration and follow-up periods, false placebo, biased or small population groups, wrong statistical analyses…). In addition, the frequent vitamin D deficiency found in cancer patients has been proposed to be a consequence and not a cause of the disease. A new round of trials encompassing many individuals, higher vitamin D doses are underway to clarify the putative beneficial effect of vitamin D in cancer prevention and/or therapy.

As an example, the first results of the VITAL trial, using 25,871 participants, follow-up period of 5.3 years and 2,000 IU vitamin D alone or in combination with omega-3 fatty acids have very recently been published. The authors concluded that supplementation with vitamin D did not result in lower incidence of invasive cancer neither cardiovascular events than placebo [65].

However, this study has several deficiencies, weaknesses and inconsistences: study population is aged (mean 67.1 years) and has high body mass index and accompanying age-related diseases and treatments. Moreover, surprisingly basal calcidiol levels were as high as 30 ng/ml. Even more importantly, the duration (5.3 years) was too short for a neoplasia such CRC that develops during 10-20 years, and the placebo group was allowed to take up to 800 IU/d vitamin D (and 20% of them admitted to take more), which could be considered as a false placebo. Still and remarkably, the authors show that if data from the first two years are not taken into account, a 25% reduction in cancer deaths was found in the vitamin D-treated group. In line with this, they conclude that a follow-up is necessary and ongoing, and that new results will be published in the next future.

Mechanistic studies in colon carcinoma cells

Already in 1992 it was reported that both calcitriol and other vitamin D metabolites inhibit cell proliferation in normal and tumoral rectum mucosa and in colon carcinoma cells [66]. Later studies have fully confirmed a variety of beneficial effects against CRC in cultured carcinoma cells and also in animal models of CRC (see for reviews[57,67,68]).

Notably, meta-analyses of epidemiological data suggest a slightly more pronounced tendency of stronger beneficial effects of vitamin D in patients with rectal tumors than with colon tumors (Figure 9).

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12

Calcitriol binds to its high affinity VDR receptor forming a stable heterodimer with the 9-cis- retinoic acid/rexinoid receptor (RXR, Figure 10). VDR belongs to the superfamily of nuclear receptors with their typical structure, a DNA binding domain (VDR binds frequently to a consensus sequence, AGGTCAnnnAGGTCA called Direct Repeat (DR)-3), a ligand binding domain and a hinge region connecting both domains. The ligand domain partially coincide with the region of ligand-dependent (carboxyterminal) transcriptional activation. It has been calculated that VDR regulates the total transcription of more than 2,000 genes directly [69,70], that vary according to the tissue/organ [71], with attributed antiproliferative, pro-apoptotic, pro- differentiation, anti-inflammatory, anti-invasion and anti-angiogenic properties [57].

According to the GTEx (Genotype-Tissue Expression) portal that compiles tissue arrays from more than 500 donors, VDR is expressed in many tissues, with its greatest expression in colon and small intestine. Six ChIP-Seq studies with VDR published so far, done in 6 different tissues, show that only 5% of the genes bound by VDR are coincident, which supports a tissue-specific response [72]. Remarkably, these VDR target genes are associated to the most relevant autoimmune diseases (multiple sclerosis, lupus, rheumatic arthritis, type 1 diabetes…), the most frequent leukemia in adults (chronic lymphocytic leukemia) and CRC [73–75].

Figure 9. CRC odds ratio in individuals with adequate levels of vitamin D stratified by tumor region: colon or rectum. Horizontal bars show the odds ratio of the studies, and the size of the points is proportional to the number of cases included in each meta-analysis (Y axis). Points on the left side of the red dashed line mean a protective effect of vitamin D against colon (up) or rectal (down) tumors.

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Figure 10. VDR

structure and attributed functions

according to Rochel and Molar (2017) [76];

Feldman el al. (2014). [57]

Calcitriol inhibits proliferation and promotes apoptosis or differentiation of human colon carcinoma cells (Figure 10) . Our group was a pioneer in describing a three-level inhibitory effect of calcitriol on the Wnt/β-catenin pathway in SW480-ADH cells (Figure 11). Calcitriol promotes VDR binding to β-catenin in the nuclei causing the reduction of the interaction of β-catenin with TCF4; 2) it induces the expression of E-cadherin, causing the accumulation of newly synthesized β-catenin at the adherens junctions and so, the reduction of nuclear b-catenin content; and 3) it upregulates the expression of DKK-1, an inhibitor of the Wnt/b-catenin pathway that impedes Wnt binding to its plasma membrane co-receptor complex [77,78]

Figure 11. Vitamin D antagonizes the Wnt/b-catenin pathway. A) Diagram showing the three antagonist mechanisms of calcitriol over the pathway. B) Phase-contrast and confocal microscopy images showing the morphological change, the induction of E-cadherin and the relocation of β-catenin from the nuclei to the plasma membrane induced by calcitriol in SW480-ADH cells. Modified from [78].

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14

Effect of calcitriol on human colonic fibroblasts

The microenvironment, niche or tumor stroma has a very important dynamic role in tumorigenesis. Tumor stroma is constituted by the extracellular matrix and diverse cell types located in the interior of the tumor mass or its proximity. Main cell types are fibroblasts (cancer- associated fibroblasts or CAF), several types of immune cells (B and T lymphocytes, macrophages/monocytes, mastocytes ...) some present in the tumor and others newly recruited from the circulation (bone marrow precursor cells); blood and lymphatic endothelial cells;

pericytes and adipocytes.

In CRC, tumor stroma and in particular CAF are considered as protumorigenic. Despite this, only one recent study from our group has analyzed the effect of calcitriol on normal fibroblasts (NAF) and CAF derived from CRC patients. Calcitriol inhibits two important protumorigenic properties of CAF: the alteration of the extracellular matrix, measured as the ability to contract collagen gels, and the induction of migratory capacity by carcinoma cells [79]. Importantly, calcitriol induces a gene signature in CAF that, like VDR expression in fibroblasts and carcinoma cells, is associated with patient good prognosis. These data show that vitamin D exerts protective effects in CRC acting not only on tumor cells but also on niche stromal fibroblasts.

Organoids

Organoids, "Method of the Year 2017" by the journal Nature Methods, are in vitro 3D organ models that constitute a "revolution" in the field as the first method to culture healthy cells from tissues without any artefactual or genetic modification. Organoids are 3D structures derived from stem cells, in which cells spontaneously self-organize into properly differentiated functional cell types [80]. They allow the study of biological processes, such as cell behavior, tissue repair and response to drugs or mutations, in an environment that mimics endogenous cell organization and organ structure. Organoids can be frozen/thawed and kept in culture indefinitely, as the population of stem cells is enriched and maintained using adequate culture media that are specific for each tissue (Figure 12).

A

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Figure 12. Organoids. A) Images of healthy rectum-derived organoids after 8 days of growth (own images). B) The diagram shows organoid types and their specific media requirements. Modified from [81]

Following the discovery of Lgr5+ stem cells in 2007 [14], a 3D culture method from fresh mouse intestinal tissue was developed for the first time (2009) [17]. A single Lgr5+ cell grew shaping spherical epithelial structures called organoids or miniguts (Figure 13A). Organoid growth required a specific complex medium, containing Wnt and R-spondin; and a extracellular matrix called Matrigel, where the cells were embedded functioning as physical and signaling support for the 3D culture. In 2011, the conditions for culturing for the first time human colon organoids were reported [82]; addition of Nicotinamide, Gastrin, PGE2 and SB202190. A minor proportion (5-6%) of cells are Lgr5+ and constitute the real stem cell population that give rise to TA cells in early phases of differentiation and to some differentiated cells.

Organoids can be obtained from complete crypts or from single Lgr5+ stem cells isolated by flow cytometry. Organoids obtained by both methods are equivalent, and in both cases are genetically stable throughout the passages (Figure 13B). The video on Figure 13C shows the first 24 h of three human colon crypts of one of our patients after being isolated and embedded in Matrigel as described in Methods. Crypt apical portion undergoes apoptosis and the crypt-bottom

B

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16

cells give rise to growing spherical/cystic human organoids that, unlike mouse small intestine organoids, do not develop the radial pseudo-crypt-shape seen in Figure 13A.

Figure 13. Organoids. A) Diagram of a mouse small intestinal organoid. Stem cells are at the bottom of the pseudo-crypts (adapted from H. Clevers Lab webpage). B) Alternative methods for intestinal organoid culture establishment. Modified from Enciclopedia Britannica. C) Video time-lapse of the evolution of human colonic crypts during 24 h after embedding the primary tissue in Matrigel (own data).

Organoids are now considered potential tools for basic cell and tissue/organ studies, developmental biology, and in regenerative medicine. As a proof-of-concept for the latter, orthotopic transplants of human organoids were performed in mice colon with induced physical damage. Once transplanted via enema in the mouse colon, the human organoids were integrated into the damaged parts regenerating functional and morphologically normal crypts (Figure 14).

Figure 14. Organoids derived from a human single LGR5+ cell are used to regenerate and repair mice damaged colonic mucosa. Scheme of the test carried out in mice that are induced to colitis with sodium dextran sulphate or DSS. On the right, end of the experiment at 25 weeks, the transplanted cells form functional and morphologically normal crypts. Adapted from Sato`s group publications in 2012 [83] and 2018 [84].

A B

C

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Currently, the organoid field is evolving towards maximizing the resemblance to the in vivo tissue structure by incorporating stromal fibroblasts, enteric nervous tissue, cells of the immune system, vascularization (angiogenesis, lymphangiogenesis) and physical forces (peristalsis…) working in vivo. As illustrated in Figure 15, it is possible to generate intestinal organoids not only from adult pluripotent cells (aPSC) but also from multipotent stem cells (embryonic or induced (iPSC)). An advantage of the latter is that they are not exclusive epithelial since are covered by an outer layer of mesenchymal cells and so, are closer to the in vivo condition.

Air-liquid interface (ALI) co-cultures preserves the epithelial-mesenchymal interaction using Transwells to grow colon sections (including all cell types) embedded in collagen and partially exposed to air, mimicking the conditions in vivo (Figure 15).

Figure 15. Methods for organoid generation. Cocultures with stroma can be achieved and are under development. Gene edition using CRISPR-Cas9 can be used in organoid cultures with various purposes, such as introducing pro-tumoral modifications. Modified from [85].

On the horizon, organoids will be the base of the so-called "organ-on-a-chip" approach that integrates cocultures of the different cell types of an organ, microfluidic perfusion and combinations of the different phases of the matter. This field has been evolving since 2010 reaching ground-breaking advances in cancer research. Future perspectives look forward the integration of the information of several "chips" of different organs and physically connect them too, trying to mimic the behavior of a complete organism (Figure 16).

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18

Figure 16. Timeline showing the development of different cancer organs-on-a-chips. [86]

Tumor organoids

Tumor organoids can grow straight-away from patient disaggregated tissues embedded in Matrigel with adequate culture media (Figure 17). LGR5+ tumor single cells give rise to organoids that can be expanded, passaged and cultured indefinitely. This also meant a revolution in the field, as there is no longer need to use mouse intermediates (PDX models).

Figure 17. Matched normal and tumor organoids can be obtained directly from patient tissues.

Modified from [87].

Despite intratumoral heterogeneity, tumor organoids, including those derived from CRC patients, fairly reproduce the pattern of mutations (at least the most relevant) existing in the tumor.

Furthermore, organoids are genetically stable during successive passes in culture [88]. In order to

Normal organoids Tumor organoids

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ensure a representation as complete as possible of tumor heterogeneity, it is recommendable to culture organoids from different areas of the tumor (T. Sato, personal communication).

A crucial aspect is to ensure that only tumoral cells and no healthy cells grow in the tumoral organoids. For CRC, this is done by removing Wnt factors from the growth media, as normal colon epithelial cells require Wnt signaling to proliferate but, in contrast, colon carcinoma cells harbour mutations that lead to the constitutive activation of the Wnt/b-catenin pathway (see above). Success in obtaining long-term growth of tumor organoids has been variably reported.

Notably, Sato et al.[89] optimized CRC tumor culture media and growth conditions (hypoxia…) managing to grow the 100% of 36 CRC organoids that included rectal tumors, serrate adenomas, and rare (neuroendocrine) CRC tumors.

Organoids as an antitumor drug assay platform

Organoid technology integrated with other technologies provide new insights into developmental processes and disease pathogenesis as well as enabling translational approaches to diagnosis and treatments. (Figure 18).

Figure 18. Organoid technology integrated with other technologies provide tools for ground- breaking applications. Modified from [90].

Hans Clevers’ group established the first biobank (“living biobank”) of CRC patient organoids [91]. After the mutational characterization of the organoids, the authors evaluated the response to a large panel of antitumor drugs with the aim of finding patterns of drug response- tumor genotype. This study was a "proof-of-concept" of the utility of organoids as a test system: it was found that organoids with mutated KRAS do not respond to EGFR inhibitors and that Nutlin was only active against organoids with a wild-type TP53 gene. These authors modified the protocol of Garnet et al. (2012) [92] to a high-throughput version to test 86 drugs on organoid cultures of 14 CRC patients. They used a robotic platform and 384 well-plates in which organoids were seeded on a layer of 2% Matrigel. However, this low percentage of Matrigel generates an almost liquid matrix in which the organoids do not grow properly and could undergo

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20

differentiation processes [93–95], which makes this protocol suitable for a first large-scale screening but does not allow the precise characterization of drug activity.

An important factor to achieve reproducibility in drug screenings is the number of organoids seeded and their size. Organoids larger than 650 μm do not allow “Cell Titer 3D”

(standard luminescent method to measure cell viability) to reach organoid inner cells and achieve a complete organoid lysis, reducing the precision of the cell viability assay. The evolution of drug screening protocols is towards size selection of the organoids prior seeding using cell strainers or image analysis software that discards wells containing organoids of undesirable sizes/morphologies.

Assays of compounds on organoids/3D cultures constitute an advantage over classic 2D systems, since their characteristics for tumor progression are closer to the situation in vivo, such as cell-cell and cell-matrix interactions, hypoxia, penetration gradient of the drugs and genetic stability, among others [96].

Towards precision medicine

As mentioned before, colon cancer patients undergo tumor resection as the first-step in their treatment and there is a window period of 6-8 weeks until the start of chemotherapy. On the contrary, rectal tumor patients are treated with radiotherapy and chemotherapy before surgery.

Current first line treatments are based on irinotecan or oxaliplatin and patient treatment decision is based on general criteria lacking prediction power over response. In case Patient “A” does not respond to an irinotecan-based treatment, successive chemotherapy treatment based on oxaliplatin will be applied. The technology of drug assays using patient-derived organoids of healthy and tumoral tissue would approach to the so-called precision medicine. According to the mutational pattern of the tumor, the "window" period provides a precious time to evaluate the patient response on both types of organoids to the different possible treatments to determine the most appropriate one. This would allow to avoid serious unnecessary side effects to the patient and save money to the health system.

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Table 2. Comparison of preclinical cancer models. Respective features were judged as best (+++), suitable (++), possible (+), not very suitable (±) or unsuitable (−). NA , not available; PDTX, patient- derived tumor xenograft (only in epithelial tumors). The immune system could be implemented by co-culturing organoids with hematopoietic cells. Reproduced from [96].

In comparison to patient-derived tumor xenografts (PDX/PDTX) which has only 30%

success in developing transplanted tumors, organoid drug assays have numerous advantages.

Tumor organoids are generated by stem cells (which according to the CSC model initiate and maintain the tumor process) and require less time, less infrastructure and so, less costs than PDX mice to reach enough “critical-mass” to allow drug assays. In addition, despite intratumoral heterogeneity, they better reproduce original mutations of the primary tumor and are genetically stable. They are not, however, an ideal system: as they do not include yet interactions with stroma, immune system or vascularization. Although its characteristics make organoids a very advantageous system, it will be necessary to rely on its optimization, a process that is underway [97].

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23

OBJECTIVES

Many epidemiological studies show an association between vitamin D deficiency and a high colorectal cancer (CRC) incidence and mortality. Moreover, data from animal models and cultures of human colon carcinoma cells and stromal fibroblasts support a protective effect of the active form of vitamin D (calcitriol) against this neoplasia. However, no studies have been reported on the putative action of calcitriol in colon stem cells, whose genetic/epigenetic alteration according to the Cancer Stem Cell (CSC) model triggers tumorigenesis and is a crucial factor in the progression of CRC.

On the other hand colon and rectal cancers are considered different clinical entities that in fact require distinct patient management, and, furthermore, neoplasias affecting different colon regions show unequal genetic alterations and response to treatments.

On the basis of these considerations, and using the "Organoid technology" as a suitable preclinical model to study healthy and diseased tissue in vitro, we aspired to shed light over some of these issues.

The Objectives of this Thesis were as follows:

1.- To establish a “living biobank” of patient-derived colorectal organoids from healthy and tumor tissue obtained from endoscopic or surgical biopsies of CRC patients.

2 To investigate whether human normal stem cells, present in the organoids and also at the bottom of the colon crypts (in fresh healthy tissue), express the nuclear vitamin D receptor (VDR).

3.- To examine whether normal and tumor colorectal organoids (stem cells and their progeny) respond to calcitriol and, if so, to study the effects of calcitriol on cell phenotype and gene expression.

4.- To analyze and compare the gene expression profiles of stem cells and their progeny in a) normal colon and rectum organoids, and b) tumor colon and rectal tumor organoids; and to investigate the response of all four types of organoid cultures to calcitriol.

5.- To set up an assay for chemotherapeutic drugs in tumor organoids generated from CRC patients.

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OBJETIVOS

Numerosos estudios epidemiológicos han asociado la deficiencia de vitamina D a una mayor incidencia y mortalidad por cáncer colorrectal (CRC). Además, los datos disponibles tanto en modelos animales como en cultivos de células humanas de carcinoma de colon y fibroblastos estromales apoyan un efecto protector de la forma activa de la vitamina D (calcitriol) frente a esta neoplasia. Sin embargo, no se ha publicado ningún estudio sobre el posible efecto del calcitriol sobre las células troncales/stem de colon, cuya alteración genética y epigenética, según el modelo de las Cancer Stem Cells (CSC), constituye el evento iniciador y probablemente impulsor de la tumorogénesis colorrectal.

Por otra parte, los cánceres de colon y de recto se consideran entidades diferentes, siendo su manejo clínico distinto, además de aceptarse diferencias entre las neoplasias originadas en las distintas regiones del colon.

Por todo ello, y valiéndonos de la “tecnología de los Organoides” como modelo válido para el estudio de tejido sano y enfermo in vitro, intentamos desentrañar algunas de estas incógnitas.

Los Objetivos de esta Tesis han sido:

1.- Generar un biobanco (living biobank) de organoides colónicos humanos a partir de células stem de biopsias de tejido sano y tumoral de pacientes con CRC obtenidas durante endoscopia o cirugía.

2.- Investigar si las células stem normales de colon humano presentes en los organoides y en el fondo de las criptas colónicas (en tejido sano fresco) expresan VDR, el receptor nuclear de la vitamina D.

3.- Investigar si los organoides derivados de células stem colónicas normales y tumorales responden al calcitriol, y en caso positivo estudiar los efectos de éste sobre el fenotipo y la expresión génica en ambos tipos de organoides.

4.- Analizar y comparar los patrones transcriptómicos de las células stem y su progenie presentes en organoides normales de colon y recto, así como en organoides tumorales de colon y recto, e investigar su respuesta de todos estos tipos de organoides al tratamiento con calcitriol.

5.- Poner a punto un sistema de ensayo de agentes quimioterápicos en organoides tumorales derivados de pacientes con CRC.

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27

MATERIALS AND METHODS

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Human samples

Human tissues were provided by IdiPAZ and Fundación Jiménez Díaz Biobanks, integrated into the Spanish Biobank Network (www.redbiobancos.es), from individuals diagnosed with colorectal cancer and subjected to surgery between 2013 and 2018. Normal tissue samples were obtained from the area distal to the tumor and the histology of the tissues was evaluated by the pathology services of La Paz University Hospital and Fundación Jiménez Díaz. All human subjects gave informed consent.

Study approval

The study complied with ethical regulations and was approved by the Ethics Committee of La Paz University Hospital (HULP-PI-1425 and HULP-PI-1639) and the Fundación Jiménez Díaz (PIC-15/2014).

Animal experiments were approved by the Ethical Committee for Animal Experimentation of the Spanish National Research Council. Vdr+/- mice were kindly provided by Prof.. MB Demay (Harvard Medical School).

Cell culture and Living Biobank

Cell lines

SW480-ADH human colon carcinoma cells were grown in DMEM supplemented with 10 % FBS (both from Thermo Fisher Scientific), 2 mM glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin.

Living Biobank

A Biobank is an organized collection of human biological material and associated information stored for one or more research purposes. The term "Living" was added since it has been possible to expand human samples in vitro [98].

Our living biobank has been created from:

1) Surgical Resection Samples . Healthy tissue to generate normal colon organoids and tumor tissue for tumor colon organoids.

2) Endoscopy Samples. Healthy colon and rectum for normal colon and normal rectum organoids and rectal tumor tissue for rectal tumor organoids.

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30

Complete crypt isolation method

Day 0 Day 1 Day 2 Day 6

Figure 19. Crypts isolated from colon pieces are embedded in Matrigel and form 3D cultures enriched in stem cells (organoids) that can be subcultured indefinitely and frozen/thawed.

3D cultures of normal and tumoral colon organoids derived from Surgical Resection Samples

Establishment and culture of normal colon organoids derived from the stem cells located in the bottom of the crypts of the colon epithelium was performed according to “complete crypt”

isolation method [98],. Healthy colon biopsies were obtained from surgical pieces (preferentially from the proximal margin). Briefly, colon mucosa biopsies were incubated with a mixture of antibiotics (Primocin, Gentamycin and Fungizone [Thermo Fisher Scientific, MA, USA]) for 30 min in rotation at room temperature (RT). Next, tissue was cut into small pieces and incubated twice with 10 mM dithiothreitol (DTT) for 5 min at RT. Samples were then transferred to 8 mM EDTA solution for 5 min at RT and 60 min in slow rotation at 4ºC. Samples were washed in PBS until complete EDTA removal and transferred to a 50 mL conical tube in fresh PBS. Colon crypts were separated from the mucosa after shaking and supernatant was centrifuged at 115xg for 5 min at RT. Crypts were washed twice in washing buffer (Advanced DMEM/F12, 10 mM HEPES, and 10 mM Glutamax) and finally pelleted crypts were embedded in Matrigel (BD Matrigel- reduced growth factor, Corning). Drops of Matrigel were seeded on pre-warmed, 48-well culture dishes. After Matrigel solidification at 37ºC “normal” culture medium was added (formulation in Table 2.1).

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