Spatial Heterogeneity of Integrins and Their Ligands in Primary Breast Tumors.

BACKGROUND: The diversity of cell-cell interactions in different regions of a tumor reflects the functional heterogeneity of cancer, which poses challenges in early diagnosis, selection of treatment strategies, and prognosis of breast cancer. Cancer cells interact with each other to form different morphological structures in the tumor and stromal host cells via integrins. The objective of this study was to characterize the morphological and spatial heterogeneity of primary breast tumors in the context of expression profiles of integrins and their ligands. METHODS: We studied spatial transcriptomics using the 10X Visium approach and the Niche Interactions and Communication Heterogeneity in Extracellular Signaling (NICHES) algorithm to map ligand-receptor signaling pathways and visualize the heterogeneity of signaling archetypes in tumor clusters. RESULTS: Cluster analysis of the expression profiles of tumor spots from the samples indicated pronounced inter-tumoral heterogeneity. Integrin-ligand functional clusters were associated with intratumoral heterogeneity, which was manifested by the presence of several morphological loci as observed in histological tumor samples. Inter-tumoral heterogeneity was manifested by a different number of functional clusters, ranging from 2 to 9 for each tumor sample. The main characteristic of these clusters was the significant predominance of non-complementary integrin subunits. Of the 42 functional integrin-ligand pairs in 21 clusters of five samples, 41 pairs occurred only once. The exception was the laminin subunit alpha-5 (LAMA5)-integrin beta 4 (ITGB4) pair, which was detected in two clusters of different samples. CONCLUSIONS: The spatial heterogeneity of integrin-ligand expression clusters in breast cancer contributes significantly to the functional heterogeneity of the tumor, which sets the stage for many scenarios of parenchymatous-stromal relationships, some of which may be effective in the emergence of metastasizing tumor seed cells. The intra- and inter-tumoral spatio-functional heterogeneity of the tumor tissue that we discovered may largely explain why it is difficult to achieve success in most patients with breast cancer using any therapeutic strategy targeting one molecule of the vast array, regardless of the importance of its pathogenetic significance.

Complex Analysis of Single-Cell RNA Sequencing Data.

Single-cell RNA sequencing (scRNA-seq) is a revolutionary tool for studying the physiology of normal and pathologically altered tissues. This approach provides information about molecular features (gene expression, mutations, chromatin accessibility, etc.) of cells, opens up the possibility to analyze the trajectories/phylogeny of cell differentiation and cell-cell interactions, and helps in discovery of new cell types and previously unexplored processes. From a clinical point of view, scRNA-seq facilitates deeper and more detailed analysis of molecular mechanisms of diseases and serves as a basis for the development of new preventive, diagnostic, and therapeutic strategies. The review describes different approaches to the analysis of scRNA-seq data, discusses the advantages and disadvantages of bioinformatics tools, provides recommendations and examples of their successful use, and suggests potential directions for improvement. We also emphasize the need for creating new protocols, including multiomics ones, for the preparation of DNA/RNA libraries of single cells with the purpose of more complete understanding of individual cells.

Angiogenesis regulators S100A4, SPARC and SPP1 correlate with macrophage infiltration and are prognostic biomarkers in colon and rectal cancers.

Introduction: Increasing evidence suggests that it is necessary to find effective and robust clinically validated prognostic biomarkers that can identify "high-risk" colorectal cancer (CRC) patients. Currently, available prognostic factors largely include clinical-pathological parameters and focus on the cancer stage at the time of diagnosis. Among cells of tumor microenvironment (TME) only Immunoscore classifier based on T lymphocytes showed high predictive value. Methods: In the present study, we performed the complex analysis of mRNA and protein expression of crucial regulators of tumor angiogenesis and tumor progression, expressed by tumor-associated macrophages (TAMs): S100A4, SPP1 and SPARC. Colon and rectal cancer patients were investigated independently and in a combined cohort (CRC). For mRNA expression, we analyzed RNA sequencing data obtained from TCGA (N=417) and GEO (N=92) cohorts of colorectal cancer patients. For protein expression, we performed IHC digital quantification of tumor tissues obtained from 197 patients with CRC treated in the Department of abdominal oncology in Clinics of Tomsk NRMC. Results: High S100A4 mRNA expression accurately predicted poor survival for patients with CRC independently of cancer type. SPARC mRNA level was independent prognostic factors for survival in colon but not in rectal cancer. SPP1 mRNA level had significant predictive value for survival in both rectal and colon cancers. Analysis of human CRC tissues revealed that S100A4, SPP1 and SPARC are expressed by stromal compartments, in particular by TAMs, and have a strong correlation with macrophage infiltration. Finally, our results indicate that chemotherapy-based treatment can change the predictive direction of S100A4 for rectal cancer patients. We found that S100A4 stromal levels were higher in patients with better response to neoadjuvant chemotherapy/chemoradiotherapy, and S100A4 mRNA levels predicted better DFS among non-responders. Discussion: These findings can help improve the prognosis of patients with CRC based on S100A4, SPP1 and SPARC expression levels.

Tumor-associated macrophages respond to chemotherapy by detrimental transcriptional reprogramming and suppressing stabilin-1 mediated clearance of EGF.

Introduction: Tumor resistance to chemotherapy and metastatic relapse account for more than 90% of cancer specific mortality. Tumor-associated macrophages (TAMs) can process chemotherapeutic agents and impair their action. Little is known about the direct effects of chemotherapy on TAMs. Methods: The effect of chemotherapeutic platinum agent cisplatin was assessed in the model system of human ex vivo TAMs. Whole-transcriptome sequencing for paired TAMs stimulated and not stimulated by cisplatin was analysed by NGS. Endocytic uptake of EGF was quantified by flow cytometry. Confocal microscopy was used to visualize stabilin-1-mediated internalization and endocytic trafficking of EGF in CHO cells expressing ectopically recombinant stabilin-1 and in stabilin-1+ TAMs. In cohort of patients with breast cancer, the effect of platinum therapy on the transcriptome of TAMs was validated, and differential expression of regulators of endocytosis was identified. Results: Here we show that chemotherapeutic agent cisplatin can initiate detrimental transcriptional and functional programs in TAMs, without significant impairment of their viability. We focused on the clearance function of TAMs that controls composition of tumor microenvironment. For the first time we demonstrated that TAMs' scavenger receptor stabilin-1 is responsible for the clearance of epidermal growth factor (EGF), a potent stimulator of tumor growth. Cisplatin suppressed both overall and EGF-specific endocytosis in TAMs by bidirectional mode: suppression of positive regulators and stimulation of negative regulators of endocytosis, with strongest effect on synaptotagmin-11 (SYT11), confirmed in patients with breast cancer. Conclusion: Our data demonstrate that synergistic action of cytostatic agents and innovative immunomodulators is required to overcome cancer therapy resistance.

Macrophage scavenger receptors: Tumor support and tumor inhibition.

Tumor-associated macrophages (TAMs) are a heterogeneous population of myeloid cells that constitute up to 50% of the cell mass of human tumors. TAMs interact with the components of the tumor microenvironment (TME) by using scavenger receptors (SRs), a large superfamily of multifunctional receptors that recognize, internalize and transport to the endosomal/lysosomal pathway apoptotic cells, cytokines, matrix molecules, lipid modified lipoproteins and other unwanted-self ligands. In our review, we summarized state-of-the art for the role of macrophage scavenger receptors in tumor development and their significance as cancer biomarkers. In this review we focused on functional activity of TAM-expressing SRs in animal models and in patients, and summarized the data for different human cancer types about the prognostic significance of TAM-expressed SRs. We discussed the role of SRs in the regulation of cancer cell biology, cell-cell and cell-matrix interaction in TME, immune status in TME, angiogenesis, and intratumoral metabolism. Targeting of tumor-promoting SRs can be a promising therapeutic approach in anti-cancer therapy. In our review we provide evidence for both tumor supporting and tumor inhibiting functions of scavenger receptors expressed on TAMs. We focused on the key differences in the prognostic and functional roles of SRs that are specific for cancer types. We highlighted perspectives for inhibition of tumor-promoting SRs in anti-cancer therapy.

PFKFB3 overexpression in monocytes of patients with colon but not rectal cancer programs pro-tumor macrophages and is indicative for higher risk of tumor relapse.

Introduction: Circulating monocytes are main source for tumor-associated macrophages (TAMs) that control tumor growth, angiogenesis, metastasis and therapy resistance. We raised the questions how monocyte programming is affected by growing tumors localized in colon and rectal sections, and how treatment onsets affect monocyte programming in the circulation. Methods: Patients with rectal cancer and colon cancer were enrolled in the study. Peripheral blood monocytes were characterized by phenotypic analysis using flow cytometry, by transcriptomic analysis using RNA sequencing and by gene expression analysis using real-time RT-PCR. Phenotypic analysis was performed with IF/confocal microscopy. Spatial transcriptomic analysis was applied using GeoMX DSP-NGS. Results: In patients with rectal cancer, increased amount of CCR2+ monocytes was indicative for the absence of both lymphatic and hematogenous metastasis. In contrast, in patients with colon cancer CD163+ monocytes were indicative for LN metastasis. NGS analysis identified tumor-specific transcriptional programming of monocytes in all CRC patients compared to healthy individuals. The key transcriptional difference between monocytes of patients with colon and rectal cancer was increased expression of PFKFB3, activator of glycolysis that is currently considered as therapy target for major solid cancers. PFKFB3-expressing monocyte-derived macrophages massively infiltrated tumor in colon. Nanostring technology identified correlation of PFKFB3 with amount and tumor-promoting properties of TAMs in colon but not in rectal cancer. PFKFB3 was indicative for tumor relapse specifically in colon cancer. Discussion: Our findings provide essential argument towards CRC definition to cover two clinically distinct cancers - colon cancer and rectal cancer, that differentially interact with innate immunity.

Effect of Early-Stage Human Breast Carcinoma on Monocyte Programming.

Circulating monocytes are a major source of tumor-associated macrophages (TAMs). TAMs in human breast cancer (BC) support primary tumor growth and metastasis. Neoadjuvant chemotherapy (NAC) is a commonly used treatment for BC patients. The absence of the response to NAC has major negative consequences for the patient: increase of tumor mass, delayed surgery, and unnecessary toxicity. We aimed to identify the effect of BC on the subpopulation content and transcriptome of circulating monocytes. We examined how monocyte phenotypes correlate with the response to NAC. The percentage of CD14-, CD16-, CD163-, and HLA-DR-expressing monocytes was quantified by flow cytometry for patients with T1-4N0-3M0 before NAC. The clinical efficacy of NAC was assessed by RECIST criteria of RECIST 1.1 and by the pathological complete response (pCR). The percentage of CD14+ and СD16+ monocytes did not differ between healthy women and BC patients and did not differ between NAC responders and non-responders. The percentage of CD163-expressing CD14lowCD16+ and CD14+CD16+ monocytes was increased in BC patients compared to healthy women (99.08% vs. 60.00%, p = 0.039, and 98.08% vs. 86.96%, p = 0.046, respectively). Quantitative immunohistology and confocal microscopy demonstrated that increased levels of CD163+ monocytes are recruited in the tumor after NAC. The percentage of CD14lowCD16+ in the total monocyte population positively correlated with the response to NAC assessed by pCR: 8.3% patients with pCR versus 2.5% without pCR (p = 0.018). Search for the specific monocyte surface markers correlating with NAC response evaluated by RECIST 1.1 revealed that patients with no response to NAC had a significantly lower amount of CD14lowCD16+HLA-DR+ cells compared to the patients with clinical response to NAC (55.12% vs. 84.62%, p = 0.005). NGS identified significant changes in the whole transcriptome of monocytes of BC patients. Regulators of inflammation and monocyte migration were upregulated, and genes responsible for the chromatin remodeling were suppressed in monocyte BC patients. In summary, our study demonstrated that presence of BC before distant metastasis is detectable, significantly effects on both monocyte phenotype and transcriptome. The most striking surface markers were CD163 for the presence of BC, and HLA-DR (CD14lowCD16+HLA-DR+) for the response to NAC.