Welcome to our Monthly Journal Club! Each month I post a paper or two that I have read and find interesting. I use this as a forum for open discussion about the paper in question. Anyone can participate in the journal club, and provide comments/critiques on the paper by leaving a comment below. I picked this month’s paper because it describes a potentially powerful new target to treat a variety of cancers using the immune system (immunotherapy). Also, I am (in part) a cancer researcher working on brain-immune interactions. Therefore, I found this paper to be very relevant to my work. The paper we are discussing is titled “CD24 signalling through macrophage Siglec-10 is a target for cancer immunotherapy” by Irving Weissman & colleagues at Stanford University.
Cancer is a complex and heterogenous disease. How can we hope to tackle this devastating illness to save lives? The classic approach of surgery followed by chemo/radiotherapy treatment is dangerous, not very effective, and can leave lasting damage that persists for the patient’s entire life. In recent years, a new strategy which harnesses the power of the immune system (immunotherapy) has gained substantial traction as a novel approach for eliminating cancer. The immune system is finely tuned to identify and kill foreign invaders (e.g., bacteria, viruses) and malignant or damaged cells (e.g., cancer cells). To do this effectively, the immune system must be able to identify ‘self’ vs ‘non-self’ in order to keep our healthy cells and tissues safe (i.e., autoimmunity). One way in which the immune system is regulated is through interactions between proteins expressed by target cells and those expressed by cells of the immune system (both lymphoid and myeloid cells). Some of these proteins enhance the immune response (e.g., MHC-II, CD28), while others drastically dampen immune activity (i.e., they are ‘don’t eat me’ signals, like PD-L1).
Tumor cells take advantage of these interactions to trick the immune system into leaving them alone, allowing them to grow and proliferate without being targeted for destruction. Irving Weissman and his colleagues were interested in identifying exactly which proteins are involved in tumor cell-immune system interactions allowing them to ‘turn off’ the immune response. From previous work on immune regulation, they knew that a protein called CD24 interacts with cells of the immune system (macrophages) through a receptor called Siglec-10 to dampen the inflammatory response. Activation of siglec-10 on macrophages by CD24 prevents the macrophage from engulfing (phagocytosis) and destroying any cell expressing CD24. Interestingly, many types of cancer express very high levels of CD24, especially ovarian cancer. Building on this information, the authors aimed to investigate the role CD24/siglec-10 signaling plays in helping cancer cells evade immune destruction.
Starting with publicly available RNA expression datasets, the authors found that nearly all tumor types they looked at expressed high amounts of CD24, and many types expressed it in higher amounts than other well described immunotherapy targets like PD-L1 and CD47 (see Figure 1 above). Additionally, tumor-associated macrophages (TAMs) express significant amounts of the binding partner (ligand) of CD24, siglec-10, and expression of CD24 is negatively associated with patient survival in ovarian and breast cancer.
To test the mechanisms that may explain these findings, the researchers started to examine how cancer cells and TAMs interact in a dish (in vitro). By using a pH sensor that glows red (pHrodo Red) and human breast cancer cells that glow green (MCF-7 GFP). When macrophages eat a tumor cell, the pH drastically changes, causing the pH sensor to glow red (see Figure 2). Using these tools, the researchers are able to quantify how many tumor cells macrophages normally eat and how many they eat when CD24 signaling is altered. Following this process over 36 h demonstrated that cells with mutated CD24 (delta-CD24) were eaten up by macrophages (i.e., destroyed) much more than cancer cells with intake CD24 signaling. Simultaneous blockade of another ‘don’t eat me’ signal (CD47) augmented this process, suggesting that these signals do not serve redundant functions and can work in tandem to achieve maximal cancer destruction. In a reciprocal experiment, knocking out (or blocking with antibodies) the binding partner of CD24 (siglec-10) also resulted in macrophages eating up many more cancer cells. This cemented the notion that CD24-siglec-10 signaling powerfully protects cancer cells from being destroyed by the immune system.
Expanding on these findings, the researchers started to look at other types of cancer, and whether manipulating CD24 could influence the destruction of cell lines from breast cancer (MCF-7), pancreatic cancer (APL1 and Panc1), or . Indeed, using a flow-cytometry approach, they were able to demonstrate that CD24 blockade, alone or in combination with CD47 blockade, drastically increased cancer cell destruction by macrophages (see Figure 3 below). Importantly, the effect was not evident in a cell line that does not normally express CD24 (U-87 MG). Moving away from cell lines, they tested whether CD24 blockade could influence the ability of macrophages to destroy primary ovarian cancer cells (that is, cells taken directly from a patient). In this case, CD24 blockade increased cancer destruction, with dual CD24 and 47 blockade being the most effective!
To move their mostly in vitro work into a more realistic in vivo model, they turned to using a laboratory mouse model of breast cancer where the cancer cells have normal (wildtype; WT) or mutated (delta-CD24) levels of CD24 expression (see Figure 4 below). By tagging the tumor cells with a gene encoding firefly luciferase (MCF-7-luc), they can track the distribution of the cancer in a living organism non-invasively using an extremely light sensitive camera. When luciferin is injected into the mice, luciferase catalyzes a reaction that results in visible light being emitted from any cell expressing luciferase (i.e., the cancer cells). This is called bioluminescence, and it can be visualized and quantified using a specialized camera.
Mice with mutated versions (that is, non-functional) of CD24 showed much less tumor growth than mice with normal expression of CD24. Additionally, depleting tumor-associated macrophages (TAMs), which eat up cancer cells, enhanced tumor growth in mice with mutated CD24. This suggests that manipulation of CD24 in a living organism (mouse) powerfully influence cancer progression, and this is largely driven by cancer cell-macrophage interactions (CD24-siglec-10). Mice that had non-functional CD24 mutations lived significantly longer than mice with normal amounts of this protein (see Fig. 4c below). When mice with functional CD24 on cancer cells were administered an antibody that blocked CD24 signaling (anti-CD24), this also significantly reduced tumor growth, although to a lesser degree than genetic mutation of CD24. This is important because it demonstrates that a single antibody treatment may be viable for treating a variety of cancers by targeting a common interaction between cancer cells and tumor associated macrophages.
Together, the data described in this paper suggest a new target for immunotherapy-mediated cancer destruction. The promise of this approach, in comparison to other immunotherapy targets, is that many different forms of cancer express high levels of CD24 (compared to popular targets like CD47 or PD-L1), it has a known binding partner (siglec-10), and anti-CD24 antibodies already exist. Additionally, CD24 isn’t expressed highly on many other types of cells that might cause significant side effects (e.g., red blood cells), so blocking this target may not have as significant side effects as other treatments. As with all of these immunotherapy treatments, significant more work is to be done to make sure that when we mess with one piece of a complex circuit, we don’t end up short circuiting the whole thing. Thanks for joining this month, leave a comment below on what you think…and as always, stay curious!