Unmasking the in vivo importance of STAT3 in multiple myeloma using three-dimensional culture and nanoparticle drug delivery system

• Author / Creator

  Huang, Yung-Hsing (Winston)

• Multiple myeloma (MM) is a hematological malignancy which is caused by aberrant accumulation of monoclonal plasma cells (PCs) within the bone marrow. The oncogenesis of MM is mediated by chromosomal aberrations as well as the tumor microenvironment in the bone marrow. Signal transducer and activator of transcription 3 (STAT3) is active in more than 50% of MM patients, and it is known to induce cell growth, survival and drug resistance in MM cells. However, activation of STAT3 in MM cells is largely dependent on exogenous soluble factors and cell adhesion. Conventional culture method is therefore not an ideal model to recapitulate the in vivo STAT3 activity of MM cells. In this thesis, I explored the importance of STAT3 in MM cells with the consideration of MM tumor microenvironment using a 3D culture model. Moreover, I aimed to develop a method for better delivery and efficacy of STAT3 inhibitor in vivo as a therapy for MM. I hypothesize that STAT3 activity in MM cells is more pronounced in close-to-in vivo tumor microenvironment, hence improvement of STAT3 inhibitor delivery by nanoparticle conjugation and anti-CD38 conjugation is a valid therapy for MM. A 3D scaffold culture model for MM cells was established for long-term culture of primary MM cells by Kirshner et al. However, the biochemical and biological effects on MM cells in this 3D model is not known. Two MM cell lines were found to have a higher level of active STAT3 (by means of phosphorylated STAT3 or pSTAT3) in the 3D culture model were compared with their counterparts in conventional culture. This elevated pSTAT3 level was dependent on the 3D environment since it decreased soon after transferring to conventional culture. STAT3 inhibition using a pharmacological agent, Stattic, significantly decreased the cell viability of MM cells and sensitized them to bortezomib in 3D culture but not in conventional culture. Using an oligonucleotide array, 3D cultured MM cells showed increased expression of several known STAT3 downstream genes implicated in oncogenesis. Primary multiple myeloma (PMM) cells harvested from patients are highly valuable resources for studying the biology of the disease and drug resistance. However, the use of these cells is limited by the fact that PMM cells are short-lived in conventional culture. The role of STAT3 and 3D culture system on prolonging the longevity of PMM cells was evaluated. Using PMM cells from patients, a significantly higher total viable PMM cell number in 3D compared to conventional culture was observed. However, the MM cell proliferation rate in the two culture systems was similar. Correlating with the high MM cell viability, higher pSTAT3 level was also observed in 3D culture. After treatment of IL6, the PMM cell viability in 3D culture was further improved. Treatment of Stattic on 3D cultured PMM cells significantly ablated their viability. On the other hand, treatment of IL6 or Stattic did not change the cell viability of conventionally cultured PMM cells. Many STAT3 inhibitors have been developed, but none of them has been approved as a cancer therapy due to their hydrophobicity and severe off-target toxicity. Lavasanifar Lab has developed a nanoparticular formation of a STAT3 inhibitor, S3I-1757 (Null-S3I-NP) for better delivery and in vivo efficacy. This nanoparticular formulation was conjugated with CD38 monoclonal antibody (denoted as CD38-S3I-NP) to further increase its MM cell targeting ability and anti-MM efficacy. CD38-S3I-NP is slightly less stable than Null-NP with a higher S3I-1757 release rate within a short time. However, CD38-conjuagred nanoparticles showed significantly higher MM cellular uptake than compared to plain nanoparticles. In keeping with this, CD38-S3I-NP resulted in a significantly lower IC50 value in two IL6-stimulated MM cell lines compared to Null-S3I-NP. CD38-S3I-NP suppressed MM tumor growth more effectively compared to Null-S3I-NP in vivo. The pSTAT3 level in the bone marrow mononuclear cells was significantly reduced in MM-bearing mice after CD38-S3I-NP treatment compared to Null-S3I-NP. These findings suggest that CD38-S3I-NP is potent anti-STAT3 agent with MM cell-targeting ability. In conclusion, this thesis reveals the essential role of STAT3 activity in maintaining the viability of MM cells in the context of 3D microenvironment. Therefore, MM cell-targeting nanoparticles with STAT3 inhibitor is believed to be a promising therapy for MM.

• Subjects / Keywords

  • STAT3
  • nanoparticle
  • multiple myeloma
  • 3D culture

• Graduation date

  Spring 2019

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-hc63-hp64

• License

  Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.