Elucidating the role of an immunomodulatory protein in cancer: From protein expression to functional characterization

Abstract

Several fundamental discoveries made over the last two decades, in the field of cancer biology, have increased our understanding of the complex tumor micro- and macroenvironments. This has shifted the current empirical cancer therapies to more rationalized treatments targeting immunomodulatory proteins. From the point of identification, a protein target undergoes several interrogations, which are necessary to truly define its druggability. Here, we outline some basic steps that can be followed for in vitro characterization of a potential immunomodulatory protein target. We describe procedures for recombinant protein expression and purification including key annotations on protein cloning, expression systems, purification strategies and protein characterization using structural and biochemical approaches. For functional characterization, we provide detailed protocols for using flow-cytometric techniques in cell lines or primary cells to study protein expression profiles, proliferation, apoptosis and cell-cycle changes. This multilevel approach can provide valuable, in-depth understanding of any protein target with potential immunomodulatory effects.

Introduction

Cancer initiation, proliferation and metastasis is a multistage process that involves alterations in the genetic material of cells, which is known as DNA mutations. Unrestrained mutagenesis of DNA disables the mechanisms that control cell growth and apoptosis and leads to conversion of normal cells into their malignant derivatives. Cancer cells are characterized by immortality (Duesberg & McCormack, 2013), genome heterogeneity (Urbach, Lupien, Karagas, & Moore, 2012), and invasiveness to other tissues, via blood or lymph nodes (Jiang et al., 2015). Current cancer therapies rely on chemotherapy, immunotherapy, hormonal therapy, surgery and radiation to control progression of cancer (Sudhakar, 2009). Despite their therapeutic impact that is highly dependent on the type and stage of cancer, these methods suffer from adverse effects due to empirical targeting approaches. Recent advances in genome sequencing (Bentley et al., 2008), along with our increased understanding of the highly complex tumor micro- and macroenvironments (Al-Zoughbi et al., 2014; Javeed & Mukhopadhyay, 2017; Sudhakar, 2009), have allowed us to identify new, promising molecular targets.

Among these targets are soluble proteins (e.g., tryptophan 2,&3-dioxygenase (TDO), indoleamine 2,3-dioxygenase-1 (IDO1), and macrophage migration inhibitory factor (MIF)), and cell surface receptors (e.g., programmed cell death protein 1 (PD-1)/programmed death ligand 1 (PD-L1), and cluster of differentiation 74 (CD74)). TDO and IDO1 are the two main enzymes that catalyze oxidation of tryptophan in the first and rate limiting step of the kynurenine pathway (Pantouris, 2012). Their immunomodulatory properties are related to tryptophan depletion (Lob, Konigsrainer, Rammensee, Opelz, & Terness, 2009; Munn et al., 1998) and the production of kynurenine pathway metabolites, such as kynurenine, that serve as endogenous tumor-promoting ligands (Opitz et al., 2011). Another immunosuppressive mechanism used by cancer cells involves upregulation of MIF, a pleiotropic protein with multiple functions that include endonuclease, enzymatic, chemokine and cytokine activities (Calandra & Roger, 2003; Wang et al., 2016). Via the latter, MIF activates the cell surface receptor CD74, promoting the survival and recruitment of inflammatory cells as well as upregulation of pro-inflammatory cytokines (Dickerhof, Schindler, Bernhagen, Kettle, & Hampton, 2015). These two mechanisms, related to the MIF induced activation of CD74, were shown to be involved in tumor survival, progression and metastasis (Simpson, Templeton, & Cross, 2012). Recently, it was demonstrated that activation of MIF/CD74 axis regulates the expression of PD-L1 in melanoma cells (Imaoka, Tanese, Masugi, Hayashi, & Sakamoto, 2019). Activation of PD-1/PD-L1 axis was found to be a critical immunosuppressive checkpoint that promotes cancer survival, proliferation and metastasis via inactivation of T cells (Alsaab et al., 2017).

Study of immunomodulatory proteins, like the ones mentioned above, offers opportunities for designing new, selective and highly potent anti-cancer therapeutics. This chapter focuses on approaches that can be followed to express, purify and characterize such proteins.

After a potential molecular target has been identified, two strategies can be followed to study the protein, at a structural and functional level:

• Expressing and purifying the recombinant protein, which can then be used for characterization using structural and biochemical methods.

• Utilizing cancer cell lines or primary cells that express the protein of interest, for evaluating the impact of small molecule therapeutics on cellular changes associated with cancer.

This chapter includes detailed protocols for recombinant protein expression and purification that can be further used for structural and biochemical characterization of the target immunomodulatory protein. The chapter also reports protocols on cellular assays using flow-cytometry. Flow-cytometry is a powerful technique that can be used to qualitatively and quantitatively address some of the key cellular changes that play a critical role in cancer progression. Using an established cancer cell line or primary cells, we provide a step-by-step protocol for protein expression analysis, proliferation, cell-cycle and apoptosis. Collectively, our protocols offer a multangular approach for the study and characterization of a protein with potential immunomodulatory function.