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Among eutherian (placental) mammals, placental embedding into the maternal endometrium exhibits great differences, from being deeply invasive (e.g., humans) to noninvasive (e.g., cattle). The degree of invasion of placental trophoblasts is positively correlated with the rate of cancer malignancy. Previously, we have shown that fibroblasts from different species offer different levels of resistance to the invading trophoblasts as well as to cancer cell invasion. Here we present a comparative genomic investigation revealing cis-regulatory elements underlying these interspecies differences in invasibility. We identify transcription factors that regulate proinvasibility and antiinvasibility genes in stromal cells. Using an in vitro invasibility assay combined with CRISPR-Cas9 gene knockout, we found that the transcription factors GATA2 and TFDP1 strongly influence the invasibility of endometrial and skin fibroblasts. This work identifies genomic mechanisms explaining species differences in stromal invasibility, paving the way to therapies targeting stromal characteristics to regulate placental invasion, wound healing, and cancer dissemination.

Abstract Background The most successful treatments for advanced RCC have been ICI-based combination therapies. However, the vast majority of patients with advanced RCC ultimately have disease progression despite ICI treatment, thus necessitating further analysis of the immunological differences in patients that respond to or are resistant to ICI therapy. CD8+ T cells display tremendous phenotypic diversity and play a critical role in anti-tumor immunity. However, it is unclear what drives different CD8+ T cell phenotypes in the RCC tumor microenvironment (TME), and how individual phenotypes impact ICI response and resistance. Methods 70 tumor samples from 63 RCC patients were collected, either before (n = 48) or after (n = 22) therapies (VEGFi, n = 9; ICI monotherapy, n = 20; ICI + ICI, n = 17; ICI + VEGFi, n = 9; others, n = 15). 11 samples were also collected from patients without tumors. RCC variants included 59 clear cell and 11 non-clear cell samples. Of these samples, 18 were labeled as clinical benefit (CB, partial or complete response as the best response) and 11 as no-clinical benefit (NCB, progressive disease as the best response). Single-cell RNA sequencing (10x Genomics) was performed on these samples in order to generate a transcriptome of the RCC tumor microenvironment (TME). Graph-based clustering was performed in order to identify cell type populations, which were then annotated using known lineage genes. Non-negative matrix factorization (NMF) was used to identify gene programs within the exhausted CD8+ T cell (Tex) population. The NicheNet algorithm was utilized to predict ligand-target interactions between macrophage cells and the Tex population. Results Within the CD8+ T cell population, Tex cells were identified through elevated expression levels of TOX, PDCD1 (PD-1), and HAVCR2 (TIM-3). NMF generated 4 gene programs within Tex cells, which expressed markers relating to immediate early genes, exhaustion/activation, tissue residency, and stress response respectively. The NMF program corresponding to tissue residency within Tex cells was associated with resistance to ICI-based therapy (P = .05); the stress program was increased in ICI response (P < .002). NicheNet identified TGF-beta (TGFB1), produced by macrophages in non-responding tumors, as the ligand with the highest regulatory potential that generated the “resistant” tissue residency program in Tex cells. It specifically identified CD69 and IL7R as notable downstream genes, which were shown to be associated with NCB through differential gene expression (P < .001). Notably, the expression of the TGF-beta receptor (TGFBR2) was significantly higher (P = .01) in Tex cells from nonresponsive tumors. To further explore the effect of TGF-beta on the tissue resident program, we sorted naïve CD8 T cells from healthy donor peripheral blood mononuclear cells, and cultured them with stimulation under hypoxia (1% O2) or normoxia, and with or without TGFb. CD69/CD103-double positive tissue-resident-like T cells were found to be significantly higher with TGFb under hypoxia (P = .02). Further, in hypoxic conditions (mimicking the RCC TME), TGF-beta also led to an increase in PD-1 expression on CD8+ T cells (P = .012). Conclusions Through single-cell RNA-seq analysis, we identify an ICI-resistance circuit whereby tumor-associated macrophages produce TGF-beta, which then leads to a tissue residency gene program in Tex cells associated with non-response to immunotherapy. Experimental validation studies demonstrated that TGF-beta and hypoxia are sufficient for the induction of a tissue residency program and PD-1 expression on CD8+ T cells. Overall, this study provides a framework for using scRNA-seq to identify mechanisms of ICI resistance in RCC, and nominates the TGF-beta axis as a potentially targetable pathway to improve CD8 T cell-mediated anti-tumor immunity.

Abstract Background Immune checkpoint blockade (ICB) therapies have revolutionized treatment for renal cell carcinoma (RCC). Despite this, only a subset of patients respond, and there remains an incomplete understanding of the cellular states that are associated with response. Defining these states may help elucidate potential mechanisms of response and assist in the development of next-generation immune therapies for RCC. Methods We utilized an existing tumor single-cell RNA sequencing (scRNA-seq) dataset from our lab of > 400 000 cells from 70 donors with RCC, with 49 of them having received ICB therapy (either anti-PD-1 alone or in different combinations). Each donor was also annotated as having benefit (complete or partial response) or non-benefit (progressive disease) after ICB therapy. Results In order to find new cell states that may impact response to ICB, we began by sub-clustering the CD4+ T cells, finding 10 clusters that existed. One cluster was strongly enriched in donors who experienced benefit after ICB therapy (Wilcoxon Test, P = .0042) and was marked by heat shock protein 70 genes (HSP70) such as HSPA6, HSPA1A, and HSPA1B. Previous work from our group has shown that many of these same HSP70 family genes are associated with response after ICB therapy in CD8+ T cells, so we hypothesized that high expression of these heat shock genes may be able to predict benefit from ICB therapy across diverse lymphocyte subsets (Kashima, ASCO, 2024). In order to test this, we made a module score of the upregulated genes from the CD4+ T cell heat shock cluster and applied it to all lymphocytes. We then calculated the correlation of the heat shock module score rank for each sub-cluster vs. the rank of how strongly each sub-cluster was associated with benefit from ICB. Strikingly, there was a nearly perfect correlation for CD4+ T cells (r = 0.89, P = .001), indicating that increased heat shock expression in CD4+ T cell clusters is associated with a better response to ICB. We repeated this test on NK and innate lymphoid cells (r = 0.77, P = .005), B cells (r = 0.67, P = .00013), and CD8+ T cells (r = 0.3, P = .325), finding similar trends each time albeit to varying extents. Next, we calculated the mean heat shock module score for each donor across all lymphocytes and calculated its difference between donors who had benefit vs. non-benefit. We found that there was a significant increase in heat shock score in donors who had benefit from ICB (Wilcoxon Test, P = .0031), and this difference was even more pronounced when only the top gene from the module (HSPA6) was used to calculate the mean difference (P = .001). To validate our findings, we turned to a pre-existing cohort of 17 donors from the HCRN GU16-260 trial where tumor scRNA-seq was available (Hugaboom et al., Cancer Discovery, 2025). After calculating the mean lymphocyte HSPA6 module score by donor, we discovered that donors who had a score greater than or equal to the median had greatly increased progression free survival (PFS) after ICB therapy (Kaplan-Meier, P = .0058; mean PFS of 14.3 months for HSPA6 high vs. 3.9 months for HSPA6 low). Conclusions We identified a heat shock signature in lymphocytes that is strongly associated with clinical benefit in RCC after ICB therapy, with the expression of the top gene (HSPA6) predicting PFS in a subset of donors from the HCRN GU16-260 clinical trial. Future studies will mechanistically test whether these genes are responsible for the therapeutic response themselves and uncover the stresses found in the tumor microenvironment that are necessary for their induction.

Abstract Background With immune checkpoint inhibitors (ICIs) now widely used as a primary treatment for metastatic RCC, understanding the composition and function of the tumor microenvironment (TME) has become more important. Numerous studies have focused on T cells to explain both treatment response and resistance, and single-cell RNA sequencing (scRNA-seq) has revealed the complex heterogeneity of T cell states. However, the contribution of myeloid cells (particularly tumor-associated macrophages (TAMs), which are known to suppress antitumor immunity) remains incompletely understood in the context of ICI therapy. In this study, we aimed to investigate TAM populations associated with ICI resistance in RCC using single-cell transcriptomic profiling. Methods We analyzed 70 tumor samples (58 clear cell and 12 non-clear cell) obtained from 63 patients with advanced RCC. The cohort included 9 untreated patients, 10 who received non-ICI systemic therapies, and 44 who were treated with ICI-based regimens. From the ICI-based therapy group, we excluded 17 patients who had stable disease and focused on 29 tumor samples from 27 patients with either pre-treatment (n = 15) or post-treatment (n = 14) samples. These patients received various ICI regimens, including mono-ICI (n = 11), ICI plus ICI (n = 11), ICI plus VEGF inhibitor (n = 6), and ICI plus IDO1 inhibitor (n = 1). We performed scRNA-seq (10x Genomics) on all samples and applied non-negative matrix factorization (NMF) to identify transcriptional programs within TAMs. We then compared these programs between responders (n = 18, complete or partial response) and non-responders (n = 11, progressive disease) based on RECIST criteria. Statistical significance was assessed using the Wilcoxon signed-rank test. Results A total of 443 337 high-quality viable cells were analyzed and classified into major cell types, including lymphoid, myeloid, tumor, endothelial, and fibroblast compartments. Within the TAM compartment, NMF uncovered 8 gene expression programs, such as “antigen presentation”, “S100A8/9 inflammation”, “stress response”, “C1Q/APOE/TREM2 signaling”, “CD163/MRC1-M2-like”, “hypoxia-related signaling”, “interferon-stimulated response”, and a distinct “LILRB/SIGLEC10” immunosuppressive program. This LILRB/SIGLEC10-enriched TAM subcluster was significantly more abundant in non-responders than in responders (P = .005). Importantly, this difference was also observed in pre-treatment samples alone (P = .014), suggesting it may be involved in primary resistance. These TAMs expressed higher expression levels of immunosuppressive LILRB1/2/3 genes, the inhibitory receptor SIGLEC10 (a recently characterized “don’t eat me” signal), and the immune checkpoint molecule VISTA, compared to other TAM subclusters (p < 2.22E-16 for each gene). Conclusions Our scRNA-seq-based analysis identified a distinct population of TAMs characterized by immunosuppressive transcriptional programs associated with poor response to ICI therapy in RCC. These findings provide insight into potential mechanisms of resistance and suggest that targeting this TAM subset may improve therapeutic efficacy. This study also demonstrates the utility of single-cell transcriptomics for uncovering key immunoregulatory populations in large clinical cohorts.

The complex behaviour of a network emerges as a product of all the interactions between its components as a single entity. The aim of system level investigations has been to identify emergent properties of a network. Identifying crucial components which are responsible for maintaining integrity of networks is essential, to understand or control them. This study presents a method to rank the participation of nodes and edges in a network using perturbation analysis to identify crucial players that contribute to the integrity of the network. The spectra of a network capture maximal features with minimal loss of information. Unlike earlier methods which evaluate perturbation in a network, based on the change in centralities or paths, the present method uses a network comparison score (Network Similarity Score) which quantifies changes at edge level to global entity level using graph spectral properties. The method is evaluated on realistic complex networks of protein structures of Muscarinic acetylcholine receptors. The important amino acid residues (nodes) and their interactions (edges) derived from the study have been correlated with experimental findings. The potential of perturbation score as a predictive tool for any real-world network is also discussed.

CD44 is an extracellular matrix receptor implicated in cancer progression. CD44 increases the invasibility of skin (SF) and endometrial stromal fibroblasts (ESF) by cancer and trophoblast cells. We reasoned that the evolution of CD44 expression can affect both, the fetal-maternal interaction through CD44 in ESF as well as vulnerability to malignant cancer through expression in SF. We studied the evolution of CD44 expression in mammalian SF and ESF and demonstrate that in the human lineage evolved higher CD44 expression. Isoform expression in cattle and human is very similar suggesting that differences in invasibility are not due to the nature of expressed isoforms. We then asked whether the concerted gene expression increase in both cell types is due to shared regulatory mechanisms or due to cell type-specific factors. Reporter gene experiments with cells and cis-regulatory elements from human and cattle show that the difference of CD44 expression is due to cis effects as well as cell type-specific trans effects. These results suggest that the concerted expression increase is likely due to selection acting on both cell types because the evolutionary change in cell type-specific factors requires selection on cell type-specific functions. This scenario implies that the malignancy enhancing effects of elevated CD44 expression in humans likely evolved as a side-effect of positive selection on a yet unidentified other function of CD44. A possible candidate is the anti-fibrotic effect of CD44 but there are no reliable data showing that humans and primates are less fibrotic than other mammals.CD44 is an extracellular matrix receptor implicated in cancer progression. CD44 increases the invasibility of skin (SF) and endometrial stromal fibroblasts (ESF) by cancer and trophoblast cells. We reasoned that the evolution of CD44 expression can affect both, the fetal-maternal interaction through CD44 in ESF as well as vulnerability to malignant cancer through expression in SF. We studied the evolution of CD44 expression in mammalian SF and ESF and demonstrate that in the human lineage evolved higher CD44 expression. Isoform expression in cattle and human is very similar suggesting that differences in invasibility are not due to the nature of expressed isoforms. We then asked whether the concerted gene expression increase in both cell types is due to shared regulatory mechanisms or due to cell type-specific factors. Reporter gene experiments with cells and cis-regulatory elements from human and cattle show that the difference of CD44 expression is due to cis effects as well as cell type-specific trans effects. These results suggest that the concerted expression increase is likely due to selection acting on both cell types because the evolutionary change in cell type-specific factors requires selection on cell type-specific functions. This scenario implies that the malignancy enhancing effects of elevated CD44 expression in humans likely evolved as a side-effect of positive selection on a yet unidentified other function of CD44. A possible candidate is the anti-fibrotic effect of CD44 but there are no reliable data showing that humans and primates are less fibrotic than other mammals.

CD44 is an extracellular matrix receptor implicated in cancer progression. CD44 increases the invasibility of skin (SF) and endometrial stromal fibroblasts (ESF) by cancer and trophoblast cells. We reasoned that the evolution of CD44 expression can affect both, the fetal–maternal interaction through CD44 in ESF as well as vulnerability to malignant cancer through expression in SF. We studied the evolution of CD44 expression in mammalian SF and ESF and demonstrate that in the human lineage evolved higher CD44 expression. Isoform expression in cattle and human is very similar suggesting that differences in invasibility are not due to the nature of expressed isoforms. We then asked whether the concerted gene expression increase in both cell types is due to shared regulatory mechanisms or due to cell type-specific factors. Reporter gene experiments with cells and cis-regulatory elements from human and cattle show that the difference of CD44 expression is due to cis effects as well as cell type-specific trans effects. These results suggest that the concerted expression increase is likely due to selection acting on both cell types because the evolutionary change in cell type-specific factors requires selection on cell type-specific functions. This scenario implies that the malignancy enhancing effects of elevated CD44 expression in humans likely evolved as a side-effect of positive selection on a yet unidentified other function of CD44. A possible candidate is the anti-fibrotic effect of CD44 but there are no reliable data showing that humans and primates are less fibrotic than other mammals.

Changes in transcriptional gene expression is a dominant mode of evolution, mostly driven by mutations at cis-regulatory regions. Mutations can affect gene expression in multiple cell types if the same cis-regulatory elements are used by different cell types. As a consequence, changes in gene expression in one cell type may be associated with similar gene expression changes in another cell type. Correlated gene expression change can explain correlated character evolution, as for instance the correlation between placental invasion and vulnerability to cancer malignancy. Here we test this hypothesis using a comparative and an experimental data set. Specifically, we investigate gene expression in dermal skin fibroblasts (SF) and uterine endometrial stomal fibroblasts (ESF). The comparative dataset consists of transcriptomes from cultured SF and ESF from 9 mammalian species. We calculated the independent phylogenetic contrasts (PIC) for each gene and cell type. We find that evolutionary changes in gene expression in SF and ESF are highly correlated, supporting the hypothesis that the correlated gene expression changes are a prevalent feature of gene expression evolution. The experimental data set derives from a SCID mouse strain that was selected for slow cancer growth which led to substantial changes in the SF compared to wild type SCID mice. We isolated SF and ESF from wild type and evolved SCID mice and compared their gene expression profiles. We find a significant correlation between the gene expression contrasts of SF and ESF, which supports the hypothesis that gene expression variation in SF and ESF is correlated. We discuss the implications of these findings for the hypothesized correlation between placental invasiveness and vulnerability to metastatic cancer.

Abstract Eutherian (placental) mammals exhibit great differences in the degree of placental invasion into the maternal endometrium, with humans being on the most invasive end. Previously, we have shown that these differences in invasiveness is largely controlled by the stromal fibroblasts of the maternal endometrium, with secondary effect on stroma of other tissues resulting in correlated differences in cancer malignancy. Here, we present a statistical investigation of the second dogma linking the phenotypic and transcriptional differences to the genomic changes across species, revealing the regulatory genomic sequence differences underlying these inter-species differences. We show that gain or loss of specific transcription factor binding site sequences are connected to the inter-species gene-expression differences in a statistically significant manner, with a particularly larger effect on stromal genes related to invasibility. We also uncover transcriptional factors differentially regulating genes related to pro- and anti- invasible property of stroma. This work extends the understanding of inter-species differences in stromal invasion to the causal genomic sequence differences paving new avenues to target stromal characteristics to regulate placental, or cancer invasion.

Evolutionary profiling has been largely limited to the nucleotide level. Using consistent proteomic methods, we quantified proteomic and phosphoproteomic layers in fibroblasts from 11 common mammalian species, with transcriptomic variability as reference. The co-variation analysis indicates that transcript and protein expression robustness across mammals remarkably follows functional role, with extracellular matrix-associated expressions being most variable, demonstrating strong transcriptome-proteome co-evolution. Interestingly, the variability control of gene expression is universal at both inter-individual and inter-species scales, but of different extent. RNA metabolism processes particularly show the higher inter-species versus inter-individual variations. Our results further uncover that while ubiquitin-proteasome system is extremely conserved in mammals, the lysosome-mediated protein degradation exhibits a remarkable variation between mammalian lineages. Additionally, the phosphosite profiles reveals phosphorylation co-evolution network independent of protein abundance. Competing Interest Statement The authors have declared no competing interest.