ENGINEERED TECHNOLOGIES TO STUDY THE ROLE OF MECHANICAL SIGNALS IN HUMAN LYMPHOMA GROWTH AND THERAPEUTIC RESPONSE
Diffuse Large B cell lymphoma (DLBCL) is the most common lymphoma representing ~30% of all B cell Non-Hodgkin lymphomas. DLBCL is a heterogeneous disease associated with a variety of clinical presentations and genetic diversity (1). The standard combination chemotherapy, Rituximab (R)-CHOP (doxorubicin, vincristine, prednisone, mechlorethamine) has been the frontline therapy for years, and still a significant percentage of DLBCL patients are not cured (2). Activated B cell-like (ABC) DLBCL is the most chemo-resistant DLBCL subtype to R-CHOP with a 5-year overall survival as low as 40% vs 80% for germinal center B cell-like (GCB) DLBCL (1, 3). A myriad of independently predictive biomarkers for resistance have been identified, including gene expression signature, stromal signatures, epigenetic silencing of specific genes, and specific somatic mutations (3, 4). But none is sufficient to predict resistance in a given patient and few are helpful in guiding the selection of targeted therapies. Therefore, new treatments and treatment-specific biomarkers are needed to improve clinical outcome of ABC-DLBCL. It is becoming increasingly evident that tumor microenvironment is an active participant in the progression and pathogenesis of lymphoma (5, 6). DLBCLs originate and reside in lymphoid tissues subjected characteristic lymphatic and vascular fluid forces, extracellular matrices, and cell-cell interactions with a variety of stromal and immune cells. In the context of ABC-DLBCL, which are the most chemoresistant subtypes, the hallmark ABC-DLBCL mutations result in constitutive activation of B cell receptor (BCR) pathways. Hence these pathways are emerging as a source of therapeutic targets for the treatment of these tumors. However to date, existing BCR pathway inhibitors such as those targeting Bruton’s tyrosine kinase (BTK) are active in a limited subset of patients and only for a short duration (few months). Therefore, there is a need to understand factors that modulate BCR. My dissertation focuses on three components of lymphoma microenvironment, namely (a) fluid shear stress and mass transport, (b) tissue stiffness, and (c) vascularization. To study the role of lymphatics-mediatedshear stress in DLBCLs, we engineered an integrated cell culture micro-reactor platform with micron-scale high resistance channels that recapitulates fluid flow velocities, pressure, and shear stresses developed in subcapsular sinuses of lymph nodes. The findings suggest that lymphatic-grade shear stress increases DLBCL cell proliferation and reduces chemotherapeutic responsiveness across DLBCL subtypes. The interplay of α4β1-integrin, CD20, and B cell receptors guides DLBCLs to respond differentially to fluid shear stress and nutrient mass transport, by altering the phosphorylation of downstream signaling pathways. The dissertation next focuses on the role of mechanical stiffness of the malignant lymphoid tissues. The DLBCLs are commonly characterized by enlargement and palpable stiffness of lymph nodes. However, there are no studies on the quantification of tissue stiffness in lymphomas and on the role, it can play in tumor progression or therapeutic response. We first determined the strain energy density of freshly isolated healthy and tumorous mouse lymph nodes, as well as lymphoma tissue from a patient using a micropipette aspiration technique, previously applied to quantify the local stiffness measurement of soft tissues (7, 8). We next developed stiffness-matched hydrogels to elucidate the role of lymphoid tissue stiffness on proliferation, phenotype, and therapeutic response of DLBCLs. The final part of the dissertation focuses on a cellular component of DLBCL microenvironment – the endothelial cells, which play an important role in angiogenesis. It is now increasingly recognized that DLBCLs are promoted by two angiogenic modes, firstly by autocrine signals via self-expressed VEGFR and VEGF, and secondly by paracrine signals of endothelial progenitors of the microenvironment (9). In this dissertation we present an engineered bio-artificial organoid platform to study the interaction between DLBCL and endothelial cells, the angiogenic component of lymphoma microenvironment. We demonstrate that two integrins α4β1 and αvβ3, expressed by both DLBCLs and endothelial cells, modulate the VEGF, as well as CD20 and B cell receptor expressed by DLCBL cells. We anticipate that our engineered technology will be useful in study of lymphoma biology, and discovery and optimization of new class of therapeutics. Key word: Lymphoma, Organoids, Integrins, Micro-reactors, Angiogenesis REFERENCES: 1. Roschewski M, Staudt LM, Wilson WH. Diffuse large B-cell lymphoma-treatment approaches in the molecular era. Nat Rev Clin Oncol. 2014;11(1):12-23. Epub 2013/11/13. doi: 10.1038/nrclinonc.2013.197. PubMed PMID: 24217204. 2. Friedberg JW. Relapsed/refractory diffuse large B-cell lymphoma. Hematology Am Soc Hematol Educ Program. 2011;2011:498-505. Epub 2011/12/14. doi: 10.1182/asheducation-2011.1.498. PubMed PMID: 22160081. 3. 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The tumour microenvironment in B cell lymphomas. Nat Rev Cancer. 2014;14(8):517-34. Epub 2014/07/11. doi: 10.1038/nrc3774. PubMed PMID: 25008267. 6. Burger JA, Wiestner A. Targeting B cell receptor signalling in cancer: preclinical and clinical advances. Nat Rev Cancer. 2018;18(3):148-67. Epub 2018/01/20. doi: 10.1038/nrc.2017.121 nrc.2017.121 [pii]. PubMed PMID: 29348577. 7. Aoki T, Ohashi T, Matsumoto T, Sato M. The pipette aspiration applied to the local stiffness measurement of soft tissues. Ann Biomed Eng. 1997;25(3):581-7. Epub 1997/05/01. PubMed PMID: 9146811. 8. Buskohl PR, Gould RA, Butcher JT. Quantification of embryonic atrioventricular valve biomechanics during morphogenesis. J Biomech. 2012;45(5):895-902. Epub 2011/12/16. doi: 10.1016/j.jbiomech.2011.11.032. PubMed PMID: 22169154; PMCID: 3535469. 9. Ruan J, Hajjar K, Rafii S, Leonard JP. Angiogenesis and antiangiogenic therapy in non-Hodgkin's lymphoma. Ann Oncol. 2009;20(3):413-24. Epub 2008/12/18. doi: 10.1093/annonc/mdn666. PubMed PMID: 19088170; PMCID: PMC2733074.
micr-bioreactor; microenvironment; Organoid; Mechanical engineering; B cell receptor; Diffuse Large B Cell Lymphoma; Integrins
Kirby, Brian; Archer, Lynden A.; Shepherd, Robert F.
Ph. D., Mechanical Engineering
Doctor of Philosophy
dissertation or thesis