By Billy Candra 
A protein, erythropoietin (EPO), identified nearly four decades ago for its primary role in stimulating red blood cell production, has now been found to play an unexpected and critical function in dampening the immune system’s response to cancer. This groundbreaking discovery, published online April 24 in the journal Science, reveals that blocking EPO’s activity can transform previously "cold" or immune-resistant liver tumors in mice into "hot" tumors, rich with immune cells capable of fighting cancer. When combined with existing immunotherapy, this approach led to the complete regression of established liver tumors in most treated mice, with animals surviving for the duration of the experiment, a stark contrast to control groups that perished within weeks.
This paradigm shift in understanding how the immune system is modulated within the tumor microenvironment holds immense promise for developing new strategies against a wide array of cancers. "This is a fundamental breakthrough in our understanding of how the immune system is turned off and on in cancer," stated Edgar Engleman, MD, PhD, a professor of pathology and medicine at Stanford University, and the senior author of the research. "I could not be more excited about this discovery, and I hope treatments that target the mechanism we uncovered will quickly move forward to human trials." David Kung-Chun Chiu, PhD, a basic life research scientist, served as the lead author of the study.
The Enigma of Erythropoietin: From Blood Booster to Immunosuppressor
Erythropoietin’s journey through medical science has been a complex one. First identified in the 1980s, EPO quickly became a cornerstone in treating various forms of anemia, particularly in patients with kidney disease or those undergoing chemotherapy. Its mechanism of action—stimulating the bone marrow to produce red blood cells—was well-understood and harnessed for therapeutic benefit. Recombinant human EPO (rhEPO) became a blockbuster drug, significantly improving the quality of life for millions by alleviating fatigue and other symptoms associated with low red blood cell counts.
However, anecdotal observations and later clinical trials began to cast a shadow on EPO’s seemingly benign role in cancer patients. By the early 2000s, studies investigating EPO use in anemic cancer patients unexpectedly reported accelerated tumor growth and reduced survival rates. This correlation was so compelling that in 2007, the U.S. Food and Drug Administration (FDA) issued a "black box warning" on EPO-stimulating agents, cautioning against their use in patients with certain cancers due to concerns about increased tumor progression and mortality.
Despite these warnings and the clear statistical link, the precise mechanism by which EPO influenced cancer growth remained elusive. The prevailing hypothesis centered on EPO’s ability to promote angiogenesis (new blood vessel formation) within tumors, thereby enhancing nutrient supply and growth. The idea that EPO might directly modulate the immune response to cancer was not widely considered. "Research from more than a decade ago has shown that giving EPO to cancer patients with anemia to stimulate red blood cell formation accelerates the growth of the tumor," Engleman explained. "Those old reports showed clearly that the more EPO or EPOR [EPO receptor] there was in tumors, the worse off the patients were. But the connection between EPO and cancer immunity was never made until now. In fact, it took a long time and a lot of experiments to convince us that EPO plays a fundamental role in blocking the immune response to cancer, because EPO is so well established as a red blood cell growth factor."
Unpacking Tumor Microenvironments: Cold vs. Hot
The research team embarked on a detailed investigation into the dynamics of liver cancer, a particularly challenging malignancy often resistant to conventional immunotherapies. Liver cancer, or hepatocellular carcinoma (HCC), is the third leading cause of cancer-related deaths globally, with rising incidence rates and limited effective treatment options for advanced stages. A significant hurdle in treating HCC, and many other solid tumors like pancreatic, colon, breast, and prostate cancers, is their classification as "cold" tumors.
"Cold" tumors are characterized by a lack of immune cell infiltration, particularly T cells, which are crucial for recognizing and destroying cancer cells. This immune-privileged state renders them largely unresponsive to immunotherapies like PD-1 inhibitors, which work by disinhibiting T cells to attack cancer. In contrast, "hot" or "inflamed" tumors are teeming with T cells and often respond well to checkpoint blockade therapies. The goal of much contemporary cancer research is to find ways to convert cold tumors into hot ones.
To explore these distinctions, Dr. Chiu meticulously developed several sophisticated mouse models of liver cancer using genome editing techniques. These models faithfully recapitulated specific genetic mutations, histological features, and responses to approved therapies observed in various human liver cancer subtypes. Tumor formation was initiated either by injecting DNA encoding liver cancer-associated proteins into the animals’ tail veins or by directly implanting liver cancer cells into the animals’ livers. This robust modeling allowed for a detailed examination of tumor development and treatment responses.
The researchers were particularly interested in the efficacy of anti-PD-1 immunotherapy, a class of drugs that target the PD-1 protein on T cells. When cancer cells bind to PD-1, they effectively "turn off" the T cells, allowing the tumor to evade immune surveillance. Anti-PD-1 therapies, such as pembrolizumab (marketed as Keytruda), have revolutionized the treatment of several cancers, including melanoma, Hodgkin’s lymphoma, and certain lung cancers, dramatically improving patient outcomes in responsive cases. However, the majority of solid tumors remain resistant.
The Unexpected Link: Hypoxia, EPO, and Immunosuppression
In their mouse models, the Stanford team observed that certain combinations of mutations led to the development of cold liver tumors that were largely ignored by the immune system and, consequently, unresponsive to anti-PD-1 treatment. These tumors exhibited minimal T cell infiltration. Conversely, other mutations resulted in hot, inflamed tumors that were rich in T cells and highly sensitive to anti-PD-1 therapy.
A critical and unexpected finding emerged when the researchers analyzed the molecular profiles of these tumors: the cold tumors displayed significantly elevated levels of EPO compared to their hot counterparts. This increase was attributed to the oxygen-poor microenvironment, a condition known as hypoxia, which is prevalent in rapidly growing, poorly vascularized tumors. Hypoxia is a well-known inducer of various proteins in cancer cells, which in turn can ramp up EPO production as a physiological response to stimulate red blood cell formation and improve oxygen delivery.
"Hypoxia in tumors has been studied for decades," Engleman noted. "It just didn’t dawn on anyone, including me, that EPO could be doing anything in this context other than serving as a red blood cell growth factor." This realization marked a pivotal moment in the research, prompting a deeper dive into EPO’s role beyond erythropoiesis.
The team then cross-referenced their findings with existing human cancer databases, confirming a strong correlation between elevated EPO levels and poorer survival rates across a range of human cancers, including those of the liver, kidney, breast, colon, and skin. This epidemiological data strongly suggested that the observations in mice might translate directly to human disease.
The Mechanism Unveiled: EPO’s Crosstalk with Macrophages
To definitively establish EPO’s role in immune suppression, the researchers conducted a series of elegant experiments. They genetically engineered cold tumor cells to be unable to produce EPO; surprisingly, these tumors transformed into hot tumors. Conversely, when hot tumors, previously sensitive to immune attack, were engineered to overexpress EPO, they thrived, becoming resistant to immune clearance.
Further exhaustive investigation pinpointed the exact cellular and molecular mechanism. In cold tumors, cancer cells produce and secrete EPO. This secreted EPO then binds to specific receptors (EPOR) located on the surface of immune cells called macrophages within the tumor microenvironment. Macrophages are versatile immune cells that can adopt different phenotypes, either promoting inflammation and immune attack (M1-like) or suppressing immunity and promoting tissue repair/tumor growth (M2-like). The binding of EPO to its receptor on macrophages was found to induce a switch in these cells towards an immunosuppressive, M2-like phenotype. These reprogrammed macrophages then actively "shoo away" cancer-killing T cells from the tumor and tamp down any residual T cell activity, effectively creating an immune desert around the tumor.
This EPO-mediated crosstalk between tumor cells and macrophages proved to be a critical determinant of immune response. The importance of this pathway was strikingly demonstrated in experiments combining the blockade of EPO signaling with anti-PD-1 therapy. In these studies, mice with cold liver tumors treated with either a control or anti-PD-1 alone did not survive beyond eight weeks post-tumor induction. However, in mice whose macrophages were genetically modified to be unable to produce the EPO receptor, 40% survived for 18 weeks, at which point the experiment was terminated. The most remarkable outcome occurred when anti-PD-1 treatment was administered to mice lacking the EPO receptor on their macrophages: all animals lived for the entire duration of the experiment, demonstrating complete tumor regression.
"It’s simple," Engleman summarized. "If you remove this EPO signaling, either by lowering the hormone levels or by blocking the receptors on the macrophages, you don’t just get a reduction in tumor growth, you get tumor regression along with sensitivity to anti-PD-1 treatment." This synergistic effect highlights the potential for a new combination therapy approach.
Broader Implications and Future Directions
The implications of this discovery are profound. The ability to convert immune-resistant cold tumors into immune-responsive hot tumors could significantly expand the reach of existing immunotherapies, offering hope to patients with cancers currently unresponsive to treatment. Liver cancer, known for its cold tumor microenvironment, stands to benefit immensely, but the correlation between elevated EPO and poorer prognosis in kidney, breast, colon, and skin cancers suggests a broader applicability.
Dr. Engleman and his colleagues are now actively designing treatments specifically targeting EPO signaling in human cancers. Several strategies are being considered. One approach involves non-specifically targeting the EPO protein itself, perhaps by inhibiting its production or function. While this could induce anemia as a side effect due to EPO’s primary role in red blood cell production, Engleman speculates that this might be an acceptable trade-off for an effective cancer therapy, especially given that anemia can be managed with transfusions or other treatments. A more refined and potentially less toxic alternative would be to selectively block the EPO receptors specifically on the surfaces of macrophages within the tumor microenvironment, leaving systemic EPO function largely intact. This precision targeting could minimize off-target effects.
The findings also provide a compelling mechanistic explanation for the 2007 FDA black box warning. It’s now clear that administering exogenous EPO to anemic cancer patients wasn’t just fueling tumor growth through angiogenesis, but actively disarming their immune system, creating a double blow that accelerated disease progression. This new understanding validates past clinical observations and points towards safer anemia management strategies in cancer patients.
The research was a collaborative effort, with contributions from researchers at the New York Blood Center and the pharmaceutical company ImmunEdge Inc. The study received funding from significant grants provided by the National Institutes of Health (R01CA262361, P01CA244114, U54CA2745115, and P01HL149626). Importantly, the lead author, Dr. Chiu, is a cofounder of ImmunEdge Inc., and Dr. Engleman is a founder, shareholder, and board member of the same company. Both are also Stanford-affiliated inventors of a patent application (PCT/US2023/063997) related to "EPO receptor agonists and antagonists," underscoring the direct translational potential and commercial interest in these findings.
"I continue to be amazed by this finding," Engleman concluded, expressing optimism tempered with scientific caution. "Not every tumor is going to respond in the same way, but I’m very optimistic that this discovery will lead to powerful new cancer therapies." The journey from a red blood cell growth factor to a key modulator of anti-cancer immunity marks a significant chapter in oncology, promising innovative approaches to overcome one of cancer’s most formidable defenses. The scientific community eagerly awaits the translation of these preclinical successes into human clinical trials, potentially ushering in a new era of cancer immunotherapy.
