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Andrea Miyahira: Hi, everyone. I'm Andrea Miyahira here at the Prostate Cancer Foundation. Joining me today is Dr. Aram Lyu from Fred Hutchinson Cancer Center. He will discuss his recent paper published in Nature, Evolution of myeloid-mediated immunotherapy resistance in prostate cancer. Dr. Lyu, thanks for joining us.

Aram Lyu: Thank you for having me, Dr. Miyahira and UroToday. Today, I'll be presenting our findings on how myeloid cell mediated immunotherapy resistance evolves during prostate cancer progression at single cell resolution. Although immune checkpoint blockade is revolutionary, patients with advanced prostate cancer are generally resistant to this therapy. This resistance is partly due to the presence of immune suppressive tumor microenvironment.

It's been well reported that myeloid cells significantly infiltrate into prostate cancer after androgen deprivation therapy, suggesting that these cells may drive immunotherapy resistance. However, efforts to target these myeloid cells broadly, such as with CSF1R antagonism, have faced clinical challenges, partly due to the heterogeneity and complexity of these cells. So our group hypothesized that if we identify distinct immunosuppressive myeloid subpopulations at the single cell level, we may be able to provide insights into increasing immunotherapy in patients.

To address this question, Dr. Fan and I performed multi-omics sequencing on patient biopsies across multiple stages. We identified a subset of macrophages expressing elevated levels of SPP1, which encodes osteopontin. These macrophages became significantly abundant during disease progression. However, their role in prostate cancer has not been rigorously investigated.

Further transcriptional analysis revealed that these macrophages exhibit elevated levels of immunosuppressive molecular programs, suggesting that they may be a key potential mediator of immunotherapy resistance in prostate cancer. To investigate in more detail, we leveraged preclinical models. We developed castration-resistant prostate cancer by engrafting Myc-CaP mouse prostate cancer, followed by degarelix treatment, performing multi-omic profiling and transcription analysis. We found SPP1 macrophages are well recapitulated in preclinical models with elevated immune suppressive transcriptional programs.

To determine whether SPP1 macrophage derived resistance to checkpoint blockade, we developed advanced prostate cancer and treated the cohort with checkpoint blockade in the presence or absence of adoptive transfer of SPP1 macrophages. We found that the introduction of these macrophages significantly increased resistance to immunotherapy, partly resulting from an increase in the frequency of exhausted CD8 T cells in the tumor microenvironment. Subsequently, transcriptional analysis and functional assays implicated adenosine signaling as an underlying mechanism.

To explore this possibility, we developed castration-resistant prostate cancer and treated the cohort with checkpoint blockade in the presence or absence of ciforadenant, which is an adenosine receptor blockade. We found that the dual treatment of PD-1 and adenosine receptor blockade significantly reduced tumor growth compared to monotherapies, suggesting that SPP1 macrophages mediate immunosuppression through adenosine signaling.

Lastly, we confirmed our findings in clinical settings. Patients with castration-resistant prostate cancer were treated with either adenosine receptor blockade alone or adenosine receptor blockade with checkpoint blockade atezolizumab. We observed that a subset of patients benefited from this dual treatment compared to monotherapies. We examined the pre-treatment biopsies from the responders and non-responders and found that there were more abundant SPP1-expressing macrophages in responders, suggesting that these macrophages may serve as predictive biomarkers for immunotherapeutic efficacy.

In conclusion, our group revealed that SPP1 macrophages increased in abundance during prostate cancer progression. And these macrophages drive immunotherapy resistance by accumulating extracellular adenosine. So blocking adenosine receptors can enhance the efficacy of immune checkpoint inhibition in patients. With that, I'd like to thank Dr. Larry Fong for his unwavering support and mentorship. I also thank Dr. Fan and former members and our collaborators, Dr. Van Allen and Miller, for their help. And lastly, I thank our funding sources, particularly Prostate Cancer Foundation, for supporting our studies. Thank you for listening.

Andrea Miyahira: So thank you so much, Dr. Lyu, for sharing this really interesting study. So in your study, you identified about 13 other tumor-infiltrating myeloid subsets in prostate cancer. Do you know what their role is and whether or not they're impacted by A2AR adenosine blockade?

Aram Lyu: In addition to SPP1-high macrophages, we also identify multiple other myeloid or macrophage subpopulations, including macrophages expressing elevated levels of CX3CR1. And these macrophages also express high levels of CD206, suggesting that these are more conventional suppressive macrophages. Interestingly, these macrophages do not express elevated levels of adenosine receptors, suggesting that they may not be targeted by adenosine receptor blockade.

However, they express elevated levels of CSF1R receptor expression, suggesting that they may be targeted by conventional CSF1R inhibition. So I believe it would be clinically important to co-target these conventional macrophages and SPP1 macrophages by blocking both adenosine receptors and CSF1R in the future.

Andrea Miyahira: OK. Thank you. And what drives SPP1 expression in the tumor-infiltrating myeloid cells? Is this subtype—are they induced from other myeloid subsets, or are they recruited specifically to tumors?

Aram Lyu: It's been reported that hypoxia can induce the levels of SPP1 in macrophages across multiple malignancies. So I believe SPP1-high macrophages can be inducible from macrophages at earlier stages. However, their developmental stages need to be rigorously investigated in the future using live trajectory analysis and functional assays. And yeah, the importance of hypoxia is partly consistent with our data.

Andrea Miyahira: OK. Thank you. And non-adenosine mechanisms such as IL-1 signaling were also contributing to immunosuppression in your model. Do you think these also need to be targeted?

Aram Lyu: Yeah, that's a great question. When we targeted adenosine receptors, we found that SPP1 macrophage-mediated immunosuppression significantly reduced. We found T-cell activity was not fully restored, suggesting that there would be additional suppressive mechanisms. And further pathway analysis and functional assays suggested that pro-inflammatory signals, including IL-1 receptor signaling, could play a key role in SPP1-high macrophage-mediated immune suppression. But at least in vitro, when we combined adenosine receptor blockade and IL-1R signaling inhibition, there was no synergistic effect. But this is in vitro, and the relevance in vivo remains to be determined more rigorously in the future.

Andrea Miyahira: OK. Thank you. And what are your next steps?

Aram Lyu: So our recent study primarily focused on the primary site, the prostate. And as we may know well, prostate cancer can metastasize into multiple organs, particularly the bone. And the bone has a very distinct myeloid landscape compared to the prostate. So as a next step, I will be exploring whether site-specific investigation of myeloid-mediated immunotherapy resistance mechanisms would be really critical for enhancing immunotherapy efficacy in prostate cancer patients.

Andrea Miyahira: Well, thank you so much, Dr. Lyu, for sharing this study with us.

Aram Lyu: Thank you for having me.