Tumors are a heterogeneous group of cells from diverse organs, ranging from stem cells and endothelial cells, to a wide range of immune cells. The plethora of secretory signals from cancer cells have numerous effects that help promote tumor growth and progression, while also perturbing the immunologic surveillance of developing tumors. Cancerous cells express their own profile of cytokines and chemokines that facilitate inflammation, cell growth, and recruitment of new blood vessels, while also recruiting accessory cell populations for their survival and immunologic avoidance. Collectively, these local changes promote the developing tumor microenvironment (TME). Multiplexed immunoassays remain the best and most complete means to study the proteomic changes within the TME, as they afford the most global view of protein changes from numerous and disparate cell populations. High-density protein expression profiling is now possible with the latest advancements in multiplex ELISA platforms, enabling detection of a diversity of novel cytokine interactions in tumor cell populations. As these unique pathways are determined, more traditional biomolecular studies can then define these networks. Multiplex ELISAs and antibody arrays therefore represent powerful tools for the identification of new cancer biomarkers, either from the local TME, or from the cancer cells themselves.
Keeping Tumors at Bay
Tumor immunosurveillance is best described as the identification and elimination of cancer cells by the immune system (Figure 1). This process is predominantly mediated by CD8+ cytotoxic T lymphocytes (CTLs), natural killer cells (NK), neutrophils, and several subtypes of effector CD4+ T cells (CD4s), with accessory roles performed by antibody producing B cells and macrophages (Mφ) amongst others (Figure 2). Effective immunosurveillance requires the innate immune system’s recognition of the tumor’s presence and the subsequent full activation and maturation of antigen presenting cell (APC) populations, namely the dendritic cell (DC) population. This maturation process increases APC surface expression of MHC-antigen complexes, increases APC endocytic sampling, upregulates cytokines that recruit T cell populations (IL-6, IL-12), and increases surface expression of T cell costimulatory ligands (CD80, CD86, ICOS). Fully mature DC populations are potent anti-tumor APCs capable of activating all forms of tumor-specific T cell populations. Activated CD8 T cells differentiate to form CTLs which have profound inflammatory and cytolytic functions, while activated effector CD4 T cells secrete cytokines that have immunostimulatory and chemotactic effects. Specifically, effector CD4 T cells develop into a T helper 1 (Th1) population which secretes IL-2 to promote CTL and further CD4 T expansion, TNF-α to inflame the site and recruit other immune cells, and IFN-γ which has anti-tumor and inflammatory functions. IFN-γ also functions to activate and drive Mφ populations into an M1 phenotype, which further produce IL-1α and IL-1β, feeding back to promote Th1 effector CD4+ polarization and reinforcing the anti-tumor immune programming. Collectively, these targeted immune responses are capable of shrinking the cancer population, but such a targeted measure can create selective pressures on those tumor cells capable of avoiding this surveillance program. The development of tumorigenesis requires the eventual subversion of immunosurveillance, a multi-step process leading to eventual escape from immunologic recognition and control.
Figure 1: Tumor-Suppressing Inflammation Model
- Human Th1/Th2/Th17 Array Q1
(detecting IFN-γ, IL-6, and IL-12 amongst others)
- Human Chemokine Array C1/G1
(detecting 38 chemokines including CXCL9 and CXCL10)
- Human Inflammation Array C1/G1 & C2/G2
(detecting IL-1α, GCSF, GM-CSF, RANTES and KC)
- Human Cytokine Array C1/G1
(detecting Several Interleukins, GRO, MCP-1 and TGF-β)
- Human Growth Factor Array C1/G1
(detecting VEGF, EGFR, TGF-β and M-CSF)
- Mouse Cytokine Array Q1
(detecting IL-1α, IL-1β, IL-6, and IL-12)
- Mouse Cytokine Array C1
(detecting IFN-γ, TNF-α, and IL-6)
Th1 lymphocytes and M1 macrophages are the primary sources of pro-inflammatory cytokines that promote cancer immunosurveillance and cytotoxicity. Their interactions are mutually reinforcing: Secretion IFN-γ by Th1 cells results in the recruitment and maintenance of M1, while IL-12 produced by M1 macrophages recruits, activates and maintains Th1cells. Secretion of MIG/CXCL9 and IP-10/CXCL10 also promotes the recruitment of Th1 cells and CTLs and inhibits angiogenesis. IL-1α, IL-1β and IL-6 form an autocrine feedback loop by stimulation of myeloid differentiation primary response gene 88 (MyD88)-mediated activation of NF-κB signaling. TNF-α, also released by the activation of NF-κB signaling, which activates APC functions of DCs and the recruitment and cytotoxic activation ofM1 macrophages, effector CD4+ T cells, and CD8+ cells, as well as the recruitment of NK cells.
Figure 2: Tumor-Supporting Immune Cell Interactions
- Human Th1/Th2/Th17 Array C1/G1
(detecting TNF-α, IL-12, IL-6, IFN-γ and IL-1β)
- Human Inflammation Array Q1 & Q2
(detecting IL-4, IL-6, IL-10, TNFα)
- Human Cytokine Array Q1000
(detecting 80 cytokines including Interleukins, IL-12p40 and p70, and IL-17)
- Mouse Inflammation Array C1/G1
(detecting IL-10, SDF-1α, IL-12p70 and IL-17p40/70)
- Mouse Inflammation Array Q1
(detecting KC, MIP-1α, MCP-1, IL-1β and IFN-γ)
- Mouse Interleukin Array Q1
(detecting G-CSF, IL-1β, IL-2, IL-4, IL-6, IL-10, and IFN-γ)
Th2 lymphocytes, M2 macrophages and MDSCs mutually reinforce the proliferation and phenotypes of one another, as well as maintaining tumor-promoting inflammation and angiogenesis. These cells, along with T regulatory lymphocytes (TREGs) suppress the activity and proliferation of tumor-suppressing cells, including Th1, M1 and cytotoxic T cells and NK cells. It should be noted that M1 &M2 macrophages can interconvert, but these phenotypes are stable as the M1 and M2 expression profiles reinforce their own macrophage phenotypes, while suppressing the other. Similarly, Th1 & Th2 lymphocytes, as well as TREG & Th17 lymphocytes tend to self-reinforce their own activation profiles and inhibit the other.
When Immunosurveillance Fails
As summarized in Figure 3, tumor escape involves many potential steps, including loss of expression or alterations of immunologically recognized tumor associated antigens (TAAs), decreased expression of NK and/or CTL recognized activation markers or increased expression of cytolytic inhibitory markers (NKG2D, MHC I, KLRG1, CTLA4), secretion of immunosuppressive cytokines (TGF-β, IL10), or recruitment of immunomodulatory cell populations to the tumor microenvironment (cancer-associated fibroblasts, monocyte-derived suppressor cells). Immunologic pressure on the growing tumor selects for increased survival of those cancer cells expressing fewer TAAs on surface MHC molecules, leading to poorer recognition by immunosurveying T cells. Reduced antigen recognition can lead to reduced cytolysis by CTLs and NK cells, but also reduces IFN-γ expression that normally promotes local inflammation and differentiation of M1 Mφs. Increased tumor cell shedding of MHC homologues can block NKG2D mediated NK cell activation, further masking the tumor cell from the innate immune system. Coupled with surface expression of CTLA4 and PD-1, molecules with known immunosuppressive functions, the tumor cell can directly interact with cancer-specific T cells and quell their cytolytic and/or cytokine-driven anti-tumor functions. Finally, reduced surface expression of the apoptotic receptors FAS and TRAIL-RI/II on the tumor surface can make the cell much more resistant to these cell death signals, especially when coupled with increased Bcl-2 or Bcl-xL levels or decreased Bim levels, together allowing the tumor to survive, evade, suppress, and even extinguish the anti-tumor response.
Figure 3: A Model of Immunoediting in Tumor Progression
- Human Apoptosis Array C1/G1
(detecting Bad, Bax, Bcl-2, Bid, Bim, Casp-3, Casp-8, and Cytochrome C)
- Human Angiogenesis Array Q1/Q2/Q3
(detecting : Angiogenin, Angiopoietin 2, TGF-β, and PLGF)
- Human Growth Factor Array Q1
(detecting SCF, VEGF-D, M-SCF and HGH)
- Human Chemokine Array C1/G1
(detecting MIP-1α, CXCL9 and CXCL10)
- Human MMP Array C1
(detecting MMP-1, -2, -3, -8, -9, -10, -13, and TIMP-1, -2, and TIMP-5)
- Mouse Angiogenesis Array C1/G1
(detecting M-CSF, G-CSF, GM-CSF, SDF-1α and IL-12)
- Mouse Cytokine Array C4/G4
(detecting bFGF, MMP2, MMP3, VEGF, IGF-1 and IGF-2)
- Mouse Cytokine Array Q3000
(detecting 160 targets including: IGF-1, IGFBP-2, VEGF-A, PLGF-2, HGF, IL-1α, IL-6, and IL-10)
Normal cells may become nascent tumors by evading tumor suppression after carcinogenic mutation and/or apoptosis that would normally result from gross chromosomal changes. Pro-inflammatory and pro-angiogenic factors can help to establish blood supply for the growing nascent tumor. Activation of the adaptive or native immune response can eliminate the nascent tumor, the tumor may remain in equilibrium as an occult tumor, or the tumor may escape immunosurveillance to create a viable tumor-supportive microenvironment. Innate and adaptive immune responses may still work to eliminate the tumor via immunosurveillance. Tumors may also metastasize to move to another location; this may be an additional mechanism of avoiding immunosurveillance by evacuation of the “hostile” TME. Green color denotes processes potentially leading to tumor eradication, while red color means promoting tumor escape and progression.
Tumor Recruitment of Accomplices
Immunoediting of cytokine signals provides another array of pathways which tumors utilize to evade and potentially escape from immunological targeting (Figure 4). Briefly, TGF-β, VEGF, angiogenic chemokines, and T helper 2 polarizing cytokines form a nexus of signals that promote tumorigenesis in certain settings. While TGF-β may indeed induce cell cycle arrest, it has many different effects within the tumor microenvironment (TME). Primed CD8 T cells receiving TGF-β signaling can block priming into fully mature CTLs, and also drive the differentiation of CD4 T cells towards a tumor-supporting T-helper 2 phenotype (Th2) or regulatory T cell poptulation (Treg), rather than a more anti-tumor Th1 phenotype. This CD4 Th2 phenotype is characterized mainly by expression of IL-4, IL-5, IL-6 and IL-10, opposite the inflammatory and anti-tumor phenotype of Th1 CD4 T cells expressing IFN-γ, TNF-α, and IL-2. Th2 polarization within the TME is further promoted by the expression of IL-10, TGF-β, and VEGF, which facilitates recruitment and differentiation of myeloid-derived suppressor cells (MDSCs), a myeloid progenitor elevated in virtually all experimental malignancies. MDSCs promote the TME in response to TGF-β signaling by expressing multiple angiogenic chemokines including CXCL-1, -2,-3, -5, -8, and -12 (amongst others), helping provide continued sustenance to the growing tumor. Increased TGF-β signaling, resulting in the conversion of CD4 T cells into a Treg poplation may promote further immunosuppressive functions, though their potential role(s) in tumorigenesis remains to be determined. VEGF is an additional pleiotropic cytokine produced inside the TME, and possesses potent immunosuppressive, inflammatory and proangiogenic capacities. The hypoxic nature of the TME results in increased HIF-1α expression which leads to increased inflammatory conditions characterized by TNFα, IL-6, and IL-8. Conveniently, these same inflammatory factors promote elevated levels of VEGF, resulting in a feedback loop of VEGF signaling, further promoting a favorable TME. VEGF is also a chemoattractant for Mφs, and with other signals surrounding the TME, these Mφs are often further primed into the MDSC population, which feeds back to promotes the immunosuppressive nature of the TME, and facilitates tumor immunosurveillance evasion.
Figure 4: Cooperativity of Cancer-Promoting Immune Cells in the TME
- Human Growth Factor Array Q1
(detecting TGF-β1, TGF-β2, TGF-β3, VEGF-A, VEGF-B and VEGF-C)
- Human Cytokine Array Q4000
(detecting CD40L, CD30, Axl, IL-4, IL-10)
- Human Chemokine Array Q1
(detecting MIF, Lymphotactin, 6Ckine, TSLP, and TARC)
- Human Angiogenesis Arrays
(detecting Angiogenin, Thrombopoietin, TGF-β, and PLGF)
- Human Cytokine Array C2000
(detecting 174 cytokines: Angiogenin, Axl, IGFBP and PDGF family members, Eotaxin, MIF, and several interleukins)
- Human MMP Array Q1
(detecting MMP-1, -2, -3, -8, -9, -10, -13, and TIMP-1, -2, and TIMP-5)
- Mouse Cytokine Array C1000/G1000
(detecting bFGF, MMP2, MMP3, IL-6, IL-10, IL-1α, VEGF, IGF-1 and IGF-2)
- Mouse Cytokine Array Q4000
(detecting 200 targets including: MMP2, MMP3, MMP10, Flt3L, VEGF, IL4, IL-10, IL-6, CD40, CD40L, and TNF-α)
In the TME, a positive feedback loop of cytokine signals that proceeds as follows: First, TGF-β, COX2, PGE2, Th2- associated inflammatory factors and proangiogenic proteins are secreted by cancer cells, CAFs and other cell types in the nascent tumor recruit Th2 lymphocytes, M2 macrophages (TAMs) andN2 neutrophils (TANs). Then, Th2 lymphocytes, TAMs and TANs secrete additional inflammatory and proangiogenic proteins that suppress maturation of DCs and proliferation and activation of cytotoxic cells. As a result, antigen presentation and cytotoxic activities plummet, practically eliminating immunosurveillance in the tumor milieu. Additionally, B cells proliferate, but are not activated, turning them into tumor-promoting BREGs. M2macrophages recruit MDSCs to the TME, further reinforcing the positive feedback loop of Th2, M2, and N2 proliferation and activation, resulting in substantial increases in tumor-promoting inflammation and concomitant angiogenesis.
Antibody Microarrays for Cancer Biomarker Discovery
Interrogation of the tumor environment’s niche of cell signals, growth factors, and cytokines, as well as the TME recruitment of accessory cell population and their cytokines, requires a global view of all these factors together. While a piecemeal approach may prove effective if the hypothesis is sufficiently narrow, a broad view of cancer will be needed for biomarker discovery and diagnostics moving forward. We believe that a type of “immunoscoring” may be warranted, whereby we can monitor the changes in a patient’s immune response, either globally or within the TME, as a routine measure of efficacy, prognosis, or disease state for cancer patients. Such a score would be heavily facilitated by the use of antibody microarrays which allow for upwards of 1000 proteins to be measured from any sample. The multiplex platform may reveal new cytokine interactions, define new complex growth factor pathways or angiogenesis mechanisms that could help open the doors to new techniques or strategies in battling this worldwide… for lack of a better word… cancer.
How Can RayBiotech Help You?
RayBiotech’s antibody array platforms and ELISAs afford you the opportunity to view the complex TME and interrogate TME-specific cytokine evasion strategies, cancer accomplice cell populations, and TME-specific signaling. The RayBio® C-Series and G-Series Arrays can semi-quantitatively detect up to 274 targets, while our Quantibody® platform can quantify up to 400 proteins targets simultaneously. Our arrays use antibody pairs against human, rhesus, mouse, rat, porcine, bovine, feline, canine, and equine cytokines. All of the aforementioned products utilize the sandwich-based ELISA technique.
Not enough targets? Consider then our L-Series Arrays, which feature a direct sample labeling technique. The L-Series allows detection of your labeled samples with single antibodies rather than a pair, allowing a much broader panel of targets to be included within 1 array. Detect up to 1000 Human proteins, 308 Mouse proteins, or 90 Rat proteins in a single sample. Additionally, we can print antibodies YOU request that are not in our current library, allowing an even more expansive array specific to your interests.
Is that too many, or do our arrays not contain some targets you are interested in? Customize it! You tell us your targets of interest, and RayBiotech will provide you with a customized array (utilizing available antibodies of your choosing).
Maybe instead you are interested in protein:protein interactions? Consider our Protein Arrays which contain up to 487 recombinant Human proteins (or 176 Mouse proteins) to capture protein interactions you are interested in.
Aren’t sure what you need, what we have, or what we are capable of? Just call us! 1-888-494-8555. You can also email us at [email protected]. We are more than happy to help you discover more.