EIKCS 2022: Macrophages and Their Role in Renal Cell Carcinoma

(UroToday.com) In the ninth session of the 2022 International Kidney Cancer Symposium (IKCS): Europe meeting focusing on basic science in renal cell carcinoma (RCC), Dr. Charles Drake presented on examining the role of macrophages in renal cell carcinoma, including two specific research foci.


First, he focused on the role of suppressive macrophages, cells that limit the immune cells’ response to cancer, in RCC. To do so, his team performed experiments to carefully understand CD45+ cells in the RCC tumor microenvironment. They relied upon specimens from 11 patients undergoing surgery for renal cell carcinoma and collected tissue from both the tumor and the adjacent, non-tumorous areas. They then performed tissue dissociation and cell sorting in order to stratify cells into four populations defined on the basis of CD45 positivity and hematopoietic origin. The authors used both single-cell RNASeq and full-spectrum flow cytometry to perform both gene expression analysis and protein activity analysis, respectively. 

Dr. Drake emphasized that the resulting single-cell data is hard to analyze. In particular, he noted that standard differential expression analysis loses a lot of information. Thus, to make better sense of the data, his group relied on a collaboration with Dr. Califano and his lab which as developed a novel approach using bioinformatics to project to a “regulon” above the level of an individual gene. In Dr. Drakes words, this approach (dubbed single-cell VIPRE protein activity inference) is like “looking through a lens”. This approach leverages gene expression of downstream targets to infer upstream protein activity and can address the so-called “dropout problem” resulting from the relatively low input of RNA from any individual cell based on scRNASeq.

While a standard analytic approach leads, in his estimation, to difficulty understanding cell types, a VIPER-based analysis demonstrated 12 distinct clusters. Notably, they found that CD8+ T cell populations were higher in tumor than in adjacent non-tumor tissue. Additionally, there were identifiable proteins in this tumor-specific CD8 cluster including TOX2, LAG3, and PDCD1.

As highlighted by the arrow in the figure below, one particular population of interest is a fairly tumor-specific macrophage population which has increased expression of TREM2, APOE, and other factors. This represents a relatively novel tumor-specific macrophage population. 

In the first of many related analyses, the authors them demonstrated that this TREM2+ cluster was associated with disease recurrence following initial surgery in an exploratory cohort of 8 patients. This has subsequently been confirmed in larger, multi-center collaborative studies. Interestingly, beyond RCC, the TREM2+ cluster has been shown to be prognostic in a variety of other cancer sites. He suggested that, while this information is not currently therapeutically actionable, it warrants further study and may represent a novel therapeutic target.

He then moved to discuss tumor infiltration T regulatory (Treg) cells. This work fits within the context of a larger, pan-cancer effort to undertake T cell profiling. In this study, the authors assessed both tumor and peripheral blood samples in order to identify tumor-related Treg and Teff cells, as well as peripheral Treg and CD4/CD8 cells. Among these naïve circulating cells, some were treated with ex vivo stimulation to act as positive controls while others were retained in their naïve state as negative controls. In taking the approach, the authors sought to identify tumor specific Treg markers. 

In defining this TI-Treg master regulator signature, Dr. Drake and his group sound to both functionally validate these candidates in vivo using pooled in vivo CRISPR screens and also to identify drugs that may invert this signature using high-throughput drug screening.

Addressing the first of these aims, he highlighted the ability of the VIPER bioinformatic analytic approach to provide nice separation of populations. As a result, they were able to identify a TI-Treg MR signature on the basis of a series of genes and transcripts that were differentially expressed. Given the use of both negative and positive controls, he emphasized that these are not standard Treg genes, but rather cancer specific Treg markers. 

Among these identified markers, TRPS1 appears to be particularly important in Treg function as a master regulator. Using tumor implantation experimentation, he showed that scrambling of TRPS1 with sgRNA results in a substantially higher proportion of tumor-free mice. 

However, the underlying biological function of TRPS1 is not known. Further, there are no conditional knockout (Flox) mice models to allow interrogation of its function. However, Dr. Drake noted that TRPS1 is expressed in “female tissues”. It is a transcriptional repressor, though without any previously known role in T cell biology. Structurally, it contains both GATA and IKAROS protein domains.

Moving to the next aim of this work, the authors sorted out Tregs from surgical specimens. They then performed progressive drug screens for decreasing Treg viability as well as drug Seq in order to identify those medications that would result in inversion of the prior signature. Notably, they identify only a few drugs that could do so, including gemcitabine plus some experimental compounds. Interestingly, dose-finding experiments demonstrated that the dose of gemcitabine could be down-titrated to levels that were one-tenth to one-twentieth the currently clinically utilized dose and still maintain the effect. Thus, ultra-low dose gemcitabine may be able to be used clinically to augment the activity of immune checkpoint inhibitors in the treatment of advanced kidney cancer. 

In conclusion, Dr. Drake highlighted the two examples of potentiation of immunotherapy in RCC, including the identification of a suppressive macrophage population in RCC with TREM2 as a potential target including therapeutically, and the identification of novel TI-Treg targets with TRPS1 representing an important one that may be druggable with very low dose gemcitabine resulting in signature inversion.


Presented by: Charles Drake, MD, PhD, VP Immuno-Oncology at The Janssen Pharmaceutical Companies of Johnson & Johnson