Towards determining the importance of bone marrow-derived factors in supporting human B acute lymphoblastic leukaemia growth in vivo

Dissertação de mestrado em Biologia Celular e Molecular, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.In the bone marrow (BM), stromal cells create special microenvironments or niches indispensable for the maintenance, proliferation and differentiation of haematopoietic stem cells (HSCs) and their derivatives, such as B cells. Moreover, evidence suggests that the BM microenvironment promotes malignant growth, including acute B lymphoblastic leukaemia (B-ALL). Currently, most of the knowledge on the interactions occurring in this malignant niche has been provided by in vitro systems and in vivo models unable to fully simulate the human BM. We set out to determine if C-X-C motif chemokine 12 (CXCL12), vascular cell adhesion molecule 1 (VCAM-1) and interleukin 7 (IL-7), stroma-derived factors, play a crucial role in B-ALL growth using a novel mouse model with subcutaneously implanted ectopic human BM niches downregulated for these factors. In vitro preliminary results not only demonstrated the biological importance of BM mesenchymal stromal cells (MSCs) and of these factors on B-ALL, but also allowed the development of functional assays to test the genetically manipulated MSCs produced. As constitutive knock down (KD) of these factors led in most cases to loss of in vivo bone forming, we generated human inducible KD MSCs for VCAM-1, IL-7 and CXCL12, and validated successful KD of VCAM-1 and IL-7 at mRNA and/or protein levels. Furthermore, to test complete ablation of our targets, we developed human knock out (KO) MSCs for CXCL12, VCAM-1 and CD44 using the CRISPR/Cas system. A decrease at the protein level of CXCL12 and VCAM-1 was observed in KO MSCs, which seemed to result in the reduction of adhesion of B-ALL cells to VCAM-1 KO MSCs. Taken together, we generated tools that allow the in vivo confirmation of our in vitro data which indicates the importance of CXCL12, VCAM-1, IL-7 for B-ALL migration, adhesion and growth. Further understanding on the communication between B-ALL cells and the BM cellular niches will contribute to the improvement of current as well as to the development of novel therapies.As células do estroma presentes na medula óssea (MO) formam microambientes ou nichos que são indispensáveis à manutenção, proliferação e diferenciação de células estaminais hematopoiéticas, assim como de células que se diferenciam a partir destas, como os linfócitos B. O microambiente da MO também favorece e promove o crescimento maligno de células cancerígenas, tal como de leucemia linfóide aguda de linfócitos B (LLA-B). A maioria das evidências experimentais atuais acerca das interações existentes neste nicho provêm de experiências in vitro e de modelos animais incapazes de simular totalmente a MO humana. O principal objetivo deste trabalho foi determinar se C-X-C motif chemokine 12 (CXCL12), vascular cell adhesion molecule 1 (VCAM-1) and interleukin 7 (IL-7), fatores derivados do estroma da MO, desempenham um papel importante no crescimento de LLA-B, utilizando um novo modelo semelhante ao sistema humano. Este modelo consiste na implantação subcutânea em murganhos imunodeficientes de nichos de MO que expressam níveis reduzidos de CXCL12, VCAM 1 e IL-7. Resultados preliminares in vitro demonstraram a importância biológica das células mesenquimais do estroma (CME) da MO e dos fatores estudados em LLA-B, e permitiram desenvolver ensaios funcionais para testar o efeito de CME geneticamente manipuladas. Como a supressão (KD) permanente destes fatores levou à perda da capacidade de formar osso in vivo, gerámos CME cujos níveis de VCAM-1, IL-7 e CXCL12 podem ser suprimidos de uma forma controlada e validámos a supressão de VCAM-1 e IL-7 ao nível do mRNA e da proteína. Para testar o efeito da supressão total destes fatores, desenvolvemos CME deficientes em VCAM-1, CXCL12 e CD44 utilizando o sistema CRISPR/Cas. Foi observado um decréscimo na expressão de CXCL12 e VCAM-1 ao nível da proteína e, sob o ponto de vista funcional, a supressão dos níveis de VCAM-1 resultou na redução da adesão de células de LLA-B. Em resumo, neste trabalho desenvolvemos ferramentas que permitem a confirmação in vivo dos nossos resultados obtidos in vitro que sugerem a importância dos fatores estromais VCAM-1, IL-7 e CXCL12 para a migração, adesão e crescimento de LLA-B. Uma melhor compreensão da comunicação entre células de LLA-B e os nichos celulares da MO permitirá a melhoria das terapias atuais e ainda o desenvolvimento de novos alvos terapêuticos

Reconstructing single-cell karyotype alterations in colorectal cancer identifies punctuated and gradual diversification patterns

Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq—a protocol that integrates live-cell imaging of tumor organoid outgrowth and whole-genome sequencing of each imaged cell to reconstruct evolving tumor cell karyotypes across consecutive cell generations. Using patient-derived colorectal cancer organoids and fresh tumor biopsies, we demonstrate that karyotype alterations of varying complexity are prevalent and can arise within a few cell generations. Sub-chromosomal acentric fragments were prone to replication and collective missegregation across consecutive cell divisions. In contrast, gross genome-wide karyotype alterations were generated in a single erroneous cell division, providing support that aneuploid tumor genomes can evolve via punctuated evolution. Mapping the temporal dynamics and patterns of karyotype diversification in cancer enables reconstructions of evolutionary paths to malignant fitness

Aneuploidy in cancer:Lessons from acute lymphoblastic leukemia

Altres ajuts: We are indebted to all the members of the laboratory of P.M. for their insights. Special thanks to Dr Isabel Granada (Hematology laboratory at Germans Trias i Pujol Hospital, Catalan Institute of Oncology, Badalona, Spain), Dr Paola Ballerini (Hopital Trousseau, Paris, France), and Drs Nassera Abermil and Chrystele Bilhou-Nabera (Saint Antoine Hospital, Paris, France) for providing the karyotype images. We thank Dr A.A. Jeyaprakash (Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh, UK) for critical revision of the MS. We thank Centres de Recerca de Catalunya (CERCA)/Generalitat de Catalunya, the Fundació Josep Carreras, and the Obra Social 'la Caixa' for their institutional support. Work in the laboratory of P.M. is supported by Spanish Ministry of Cience and Innovation, the European Research Council (CoG-2014-646903 and PoC-2018-811220), the Spanish Ministry of Economy and Competitiveness , the Fundación Uno entre Cienmil, and the Fundación Leo Messi. F.S. is supported by Instituto de Salud Carlos III, the Spanish Ministry of Economy and Competitiveness and (GRC). We also thank the Wellcome Trust for supporting M.A.A. through a Senior Research Fellowship to A.A. Jeyaprakash (202811). P.M. is an investigator of the Spanish Cell Therapy Cooperative Network (TERCEL).Aneuploidy, the gain or loss of chromosomes in a cell, is a hallmark of cancer. Although our understanding of the contribution of aneuploidy to cancer initiation and progression is incomplete, significant progress has been made in uncovering the cellular consequences of aneuploidy and how aneuploid cancer cells self-adapt to promote tumorigenesis. Aneuploidy is physiologically associated with significant cellular stress but, paradoxically, it favors tumor progression. Although more common in solid tumors, different forms of aneuploidy represent the initiating oncogenic lesion in patients with B cell acute lymphoblastic leukemia (B-ALL), making B-ALL an excellent model for studying the role of aneuploidy in tumorigenesis. We review the molecular mechanisms underlying aneuploidy and discuss its contributions to B-ALL initiation and progression

Live imaging of cell division in 3D stem-cell organoid cultures

Examining cell behavior in its correct tissue context is a major challenge in cell biology. The recent development of mammalian stem cell-based organoid cultures offers exciting opportunities to visualize dynamic cellular events in a 3D tissue-like setting. We describe here an approach for live imaging of cell division processes in intestinal organoid cultures derived from human and mouse adult stem cells. These approaches can be extended to the analysis of cellular events in diseased tissue, such as patient-derived tumor organoids

Live imaging of cell division in 3D stem-cell organoid cultures

Examining cell behavior in its correct tissue context is a major challenge in cell biology. The recent development of mammalian stem cell-based organoid cultures offers exciting opportunities to visualize dynamic cellular events in a 3D tissue-like setting. We describe here an approach for live imaging of cell division processes in intestinal organoid cultures derived from human and mouse adult stem cells. These approaches can be extended to the analysis of cellular events in diseased tissue, such as patient-derived tumor organoids

Organoids from colorectal peritoneal metastases as a platform for improving hyperthermic intraperitoneal chemotherapy

BACKGROUND: Patients with peritoneal metastases from colorectal cancer have a poor prognosis. If the intraperitoneal tumour load is limited, patients may be eligible for cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (HIPEC). This treatment has improved overall survival, but recurrence rates are high. The aim of this study was to create a preclinical platform for the development of more effective intraperitoneal chemotherapy strategies. METHODS: Using organoid technology, five tumour cultures were generated from malignant ascites and resected peritoneal metastases. These were used in an in vitro HIPEC model to assess sensitivity to mitomycin C (MMC) and oxaliplatin, the drugs used most commonly in HIPEC. The model was also used to test a rational combination treatment involving MMC and inhibitors of the checkpoint kinase ATR. RESULTS: MMC was more effective in eliminating peritoneal metastasis-derived organoids than oxaliplatin at clinically relevant concentrations. However, the drug concentrations required to eliminate 50 per cent of the tumour cells (IC50) were higher than the median clinical dose in two of five organoid lines for MMC, and all five lines for oxaliplatin, indicating a general resistance to monotherapy. ATR inhibition increased the sensitivity of all peritoneal metastasis-derived organoids to MMC, as the IC50 decreased 2·6-12·4-fold to well below concentrations commonly attained in clinical practice. Live-cell imaging and flow cytometric analysis showed that ATR inhibition did not release cells from MMC-induced cell cycle arrest, but caused increased replication stress and accelerated cell death. CONCLUSION: Peritoneal metastasis-derived organoids can be used to evaluate existing HIPEC regimens on an individual-patient level and for development of more effective treatment strategies. Surgical relevance Cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (HIPEC) has improved prognosis of patients with peritoneal metastases from colorectal cancer, but disease recurrence is common. More effective and personalized HIPEC is urgently needed. Organoid technology is frequently used for drug screens, as patient-derived organoids can accurately predict clinical therapeutic response in vitro. A panel of organoids was established from peritoneal metastases from colorectal cancer and used to develop a model for testing HIPEC regimens in vitro. Patient-derived organoids differed in sensitivity to commonly used chemotherapeutics, in line with variable clinical outcomes following cytoreductive surgery-HIPEC. Combining MMC with an ATR inhibitor improved the efficacy of MMC. Peritoneal metastasis-derived organoids can be used as a platform to test novel (combination) strategies that increase HIPEC efficacy. In the future, organoids could be used to select patent-tailored HIPEC regimens

Organoids from colorectal peritoneal metastases as a platform for improving hyperthermic intraperitoneal chemotherapy

BACKGROUND: Patients with peritoneal metastases from colorectal cancer have a poor prognosis. If the intraperitoneal tumour load is limited, patients may be eligible for cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (HIPEC). This treatment has improved overall survival, but recurrence rates are high. The aim of this study was to create a preclinical platform for the development of more effective intraperitoneal chemotherapy strategies. METHODS: Using organoid technology, five tumour cultures were generated from malignant ascites and resected peritoneal metastases. These were used in an in vitro HIPEC model to assess sensitivity to mitomycin C (MMC) and oxaliplatin, the drugs used most commonly in HIPEC. The model was also used to test a rational combination treatment involving MMC and inhibitors of the checkpoint kinase ATR. RESULTS: MMC was more effective in eliminating peritoneal metastasis-derived organoids than oxaliplatin at clinically relevant concentrations. However, the drug concentrations required to eliminate 50 per cent of the tumour cells (IC50) were higher than the median clinical dose in two of five organoid lines for MMC, and all five lines for oxaliplatin, indicating a general resistance to monotherapy. ATR inhibition increased the sensitivity of all peritoneal metastasis-derived organoids to MMC, as the IC50 decreased 2·6-12·4-fold to well below concentrations commonly attained in clinical practice. Live-cell imaging and flow cytometric analysis showed that ATR inhibition did not release cells from MMC-induced cell cycle arrest, but caused increased replication stress and accelerated cell death. CONCLUSION: Peritoneal metastasis-derived organoids can be used to evaluate existing HIPEC regimens on an individual-patient level and for development of more effective treatment strategies. Surgical relevance Cytoreductive surgery followed by hyperthermic intraperitoneal chemotherapy (HIPEC) has improved prognosis of patients with peritoneal metastases from colorectal cancer, but disease recurrence is common. More effective and personalized HIPEC is urgently needed. Organoid technology is frequently used for drug screens, as patient-derived organoids can accurately predict clinical therapeutic response in vitro. A panel of organoids was established from peritoneal metastases from colorectal cancer and used to develop a model for testing HIPEC regimens in vitro. Patient-derived organoids differed in sensitivity to commonly used chemotherapeutics, in line with variable clinical outcomes following cytoreductive surgery-HIPEC. Combining MMC with an ATR inhibitor improved the efficacy of MMC. Peritoneal metastasis-derived organoids can be used as a platform to test novel (combination) strategies that increase HIPEC efficacy. In the future, organoids could be used to select patent-tailored HIPEC regimens

Reconstructing single-cell karyotype alterations in colorectal cancer identifies punctuated and gradual diversification patterns

Central to tumor evolution is the generation of genetic diversity. However, the extent and patterns by which de novo karyotype alterations emerge and propagate within human tumors are not well understood, especially at single-cell resolution. Here, we present 3D Live-Seq—a protocol that integrates live-cell imaging of tumor organoid outgrowth and whole-genome sequencing of each imaged cell to reconstruct evolving tumor cell karyotypes across consecutive cell generations. Using patient-derived colorectal cancer organoids and fresh tumor biopsies, we demonstrate that karyotype alterations of varying complexity are prevalent and can arise within a few cell generations. Sub-chromosomal acentric fragments were prone to replication and collective missegregation across consecutive cell divisions. In contrast, gross genome-wide karyotype alterations were generated in a single erroneous cell division, providing support that aneuploid tumor genomes can evolve via punctuated evolution. Mapping the temporal dynamics and patterns of karyotype diversification in cancer enables reconstructions of evolutionary paths to malignant fitness

Ongoing chromosomal instability and karyotype evolution in human colorectal cancer organoids

Chromosome segregation errors cause aneuploidy and genomic heterogeneity, which are hallmarks of cancer in humans. A persistent high frequency of these errors (chromosomal instability (CIN)) is predicted to profoundly impact tumor evolution and therapy response. It is unknown, however, how prevalent CIN is in human tumors. Using three-dimensional live-cell imaging of patient-derived tumor organoids (tumor PDOs), we show that CIN is widespread in colorectal carcinomas regardless of background genetic alterations, including microsatellite instability. Cell-fate tracking showed that, although mitotic errors are frequently followed by cell death, some tumor PDOs are largely insensitive to mitotic errors. Single-cell karyotype sequencing confirmed heterogeneity of copy number alterations in tumor PDOs and showed that monoclonal lines evolved novel karyo-types over time in vitro. We conclude that ongoing CIN is common in colorectal cancer organoids, and propose that CIN levels and the tolerance for mitotic errors shape aneuploidy landscapes and karyotype heterogeneity