By Bekah McBride
When illness strikes, white blood cells are the first cells on the scene. Much like a team of superheroes they each complete their specialized task, engulfing bacteria, controlling the immune response, and cleaning up dead or damaged cells. Leading the charge, is the macrophage, which literally translates to “big eater”. These white blood cells destroy illnesses, recruit other immune cells, and clean up the aftermath. Given their incredible power to heal us, researchers at the University of Wisconsin–Madison are exploring ways that these cells can be used to fight tumors through immunotherapy.
“We know that macrophages like to naturally hang out at the site of solid tumors,” said UW–Madison postdoctoral research associate, Portia Smith, PhD. “So, instead of fighting the biology of cancer, we wanted to work with it.”
To super charge these superhero cells, Smith, who completed this work in the lab of UW–Madison Stem Cell and Regenerative Medicine Center (SCRMC) member and Professor of Pathology and Laboratory Medicine, Igor I Slukvin, MD, PhD, utilized human induced pluripotent stem cells (iPSCs) to develop a more effective cellular therapy for the treatment of solid tumors, including ovarian cancer and pediatric neuroblastoma.
Utilizing CRISPR/Cas9 gene editing technology and iPSCs, which are stem cells that can be created from nearly any type of somatic cell in the body, such as skin or blood cells, Smith was able to generate genetically modified macrophages to target the inhibitory pathways within the tumor microenvironment. Unlike donor-derived macrophages, iPSCs can be expanded indefinitely in culture, providing a virtually unlimited and renewable source of gene-edited macrophages for cellular therapies without the need for repeated donor cell collection. This modification makes the macrophages even more powerful, by allowing them to not only detect cancerous or dying cells but program them to kill the solid tumor.
To do this, Smith used a highly specified protocol, developed in the Slukvin Lab, called ‘in vitro hematopoiesis’, that essentially allows the team to generate blood cells [and macrophages] from stem cells in a petri dish. The team was then able to use these iPSC-derived macrophages (iMacs) to target the “eat me”/ “don’t eat me” molecular pathways that cancer cells exploit to avoid being “eaten” or phagocytosed by macrophages.
“Macrophages have the capacity to engulf and destroy foreign pathogens, damaged cells, cellular debris, and even cancer cells through a process called phagocytosis, and during this process there are checkpoints” says Smith.
These checkpoints are a way for the macrophages to ensure they are only “eating” the bad or damaged cells, and spare normal and healthy cells from phagocytosis. However, tumor cells find multiple ways to evade the immune system, including using these checkpoints to convince the immune system that they are healthy cells (i.e. “don’t eat me”). The cancer cells manipulate the immune system by upregulating, or increasing, “don’t eat me receptors”, one of which is known as CD47. To evade this mechanism used by tumors, Smith and the Slukvin Lab learned that macrophages have a receptor called SIRPalpha (SIRPα), which recognizes CD47. So, although macrophages can access the site of the solid tumor very easily, they aren’t able to destroy the cancer cells due to the upregulation of CD47 “don’t eat me” receptor.
“We wanted to target the ablation, or the knockout, of the SIRPα gene to take off the “brake” and allow the macrophages to reach their full potential in targeting the cancer cells,” says Smith.
By knocking out CD47, the team found that they can successfully generate enhanced anti-tumor activities when paired with a tumor-directed accelerant. The first accelerant was monoclonal antibody therapy, which is FDA approved and can target HER2, a tumor associated antigen (i.e. foreign material targeted for destruction) that is highly expressed on ovarian cancer.
“To target the cancer cells, we use these SIRPα-knockout macrophages as a combination therapy,” says Smith. “First, we take the “brake” off the macrophages by knocking-out SIRPα, then add the “gas” or accelerant by using tumor-targeting monoclonal antibody and found an enhanced anti-tumor effect.”
The second method used chimeric antigen receptor (CAR) cells. This time, instead of adding the antibody, the receptor that recognized the tumor was attached to the cell itself.
“Both forms of those accelerants provided anti-tumor efficacy when combined with the SIRPα knock-out,” says Smith. “Our most important finding was that knocking-out SIRPα not only enhanced anti-tumor capacity, but it also protected the macrophages against exhaustion, a very particular type of phagocytic-related exhaustion, called hypophagia.”
The team discovered that when they knocked out SIRPα, the cells did not experience hypophagia and could continue “eating” even after multiple exposures to the cancer.
“Exhaustion has been mostly studied in T cells, so our understanding of macrophage exhaustion, or hypophagia, is very limited and new to the field.” says Smith. “So, when we found that SIRPα is a key regulator macrophage exhaustion, and that we can manipulate and control that that type of function of the macrophages through ablation of SIRPα, we had a reason to be excited.”
Smith continued, “This is such an important finding because macrophages exist in every tissue within your body and in every type of cancer. If we can understand more about their biology and how they operate in the face of tumors, then we can figure out better ways to target them. This will help in the fight against any type of cancer.”
While this is still a pre-clinical model, meaning it has not yet been tested in large animal models or humans, gaining a better understanding of hypophagia, and the impact of knocking out SIRPα, is a significant step towards using these superhero cells to improve therapies.
“The main takeaway is that SIRPα -KO macrophages can recognize and kill cancer efficiently in vitro, but they don’t tend to stick around for a long timeusing in vivo preclinical models of solid tumors, leading to diminished results. Therefore, we need to investigate additional ways to enhance their capacity to have a clinical benefit,” says Smith.
Right now, Smith is working in the lab of Christian Capitini, MD, a Stem Cell and Regenerative Medicine Center member and the Director of the Carbone Cancer Center, through a Robert Connor Dawes (RCD) Foundation fellowship, studying a combinational therapy for tumors using the CAR macrophages and the CAR-T cells.
“CAR-T cells have the capacity to persist for a long time in the body, but a hard time penetrating within solid tumors. Whereas macrophages are the opposite.” says Smith. “So, using both advantages of each type of immune cell, we hope using both CAR macrophages and CAR T cells against hard-to-treat solid tumors could possibly lead to a greater benefit overall.”
While additional research is required before this will reach the clinic, Smith says this study is an important piece of the puzzle.
It goes without saying that most people have been affected by cancer, either personally or through a loved one. It’s a devastating disease in its many forms and a very large financial burden. As researchers, we’re always aiming to generate therapies that are both effective at killing cancer and more accessible to patients,” says Smith. “Funding this type of research is critical; even if we don’t get all the answers now, every study adds to the vast knowledgebase of science that builds upon itself over decades. This particular study was important for better understanding how macrophages interact with cancer cells and how we can rewire them to resist exhaustion by knocking-out SIRPα. Though there may be years of work before me, it’s inspiring knowing that this may eventually impact patients that really need these therapies.”