A Single-Cell Transcriptional Roadmap of the Mouse and Human Lymph Node Lymphatic Vasculature

Single-cell transcriptomics promise to revolutionize our understanding of the vasculature. Emerging computational methods applied to high-dimensional single-cell data allow integration of results between samples and species and illuminate the diversity and underlying developmental and architectural organization of cell populations. Here, we illustrate these methods in the analysis of mouse lymph node (LN) lymphatic endothelial cells (LEC) at single-cell resolution. Clustering identifies five well-delineated subsets, including two medullary sinus subsets not previously recognized as distinct. Nearest neighbor alignments in trajectory space position the major subsets in a sequence that recapitulates the known features and suggests novel features of LN lymphatic organization, providing a transcriptional map of the lymphatic endothelial niches and of the transitions between them. Differences in gene expression reveal specialized programs for (1) subcapsular ceiling endothelial interactions with the capsule connective tissue and cells; (2) subcapsular floor regulation of lymph borne cell entry into the LN parenchyma and antigen presentation; and (3) pathogen interactions and (4) LN remodeling in distinct medullary subsets. LEC of the subcapsular sinus floor and medulla, which represent major sites of cell entry and exit from the LN parenchyma respectively, respond robustly to oxazolone inflammation challenge with enriched signaling pathways that converge on both innate and adaptive immune responses. Integration of mouse and human single-cell profiles reveals a conserved cross-species pattern of lymphatic vascular niches and gene expression, as well as specialized human subsets and genes unique to each species. The examples provided demonstrate the power of single-cell analysis in elucidating endothelial cell heterogeneity, vascular organization, and endothelial cell responses. We discuss the findings from the perspective of LEC functions in relation to niche formations in the unique stromal and highly immunological environment of the LN

Targeting OGG1 arrests cancer cell proliferation by inducing replication stress

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment

The role of the lymph node stroma in cancer and aging

The function of the lymph node (LN) stroma is crucial for the induction of an adaptive immune response. The stroma includes lymphatic vessels, high endothelial venules (HEVs) and mesenchymal cells (MCs). In this thesis, I have been investigating the role of the LN stroma in cancer and aging. In Paper I, we demonstrate that LNs draining invasive breast cancer present remodeling of the HEVs and dysregulation of the perivascular MCs. Dilation of HEVs is correlating with inhibition of normal lymphocyte recruitment due to perivascular changes in the expression of molecular cues necessary for chemotaxis. As a follow up of Paper I, we created an automatic method for image analysis of HEVs by using artificial intelligence and deep learning. In paper II, we validate the HEV-finder that we suggest is a good automatic image analysis tool to study HEV remodeling in different subtypes of cancer and to perform survival studies in larger patient cohorts. In paper III, we present a mapping of the lymphatic endothelial cells (LECs) of the mouse and human LNs by using single-cell RNA sequencing and present a new LEC subset called PTX3-LECs. We demonstrate that both in mouse and human LNs there are four LEC subsets, forming the subcapsular and medullary sinuses where immune cells enter and exit the LN, respectively. Our mapping is a great tool for further investigation of different subsets and their specific genes in translational studies of homeostasis or disease. In paper IV, we are focusing on LN lipomatosis, which is an age-related phenomenon where the normal LN parenchyma is replaced by adipose tissue. Our data support that LN lipomatosis is developing in the medulla of the LN due to transdifferentiation of medullary MCs into adipocytes. We found that it is causing remodeling of the HEVs and loss of medullary lymphatic vessels, affecting both the entry and exit of lymphocytes. Based on our findings, we suggest that LN lipomatosis is a contributing factor in immunodeficiency in elderly. In paper V, we created a new transplantable breast cancer model in mouse to study the effect of LN metastasis by inducing the expression of the chemokine receptor CCR7 in the mammary carcinoma cell line EO771. The new model will allow us to study the effect of LN metastasis on the anti-tumor immunity and response to therapy. In summary, my thesis shed light on the importance of a functioning LN stroma in both cancer and aging and provides new tools for image analysis, translational studies of the LN stroma and models for LN metastasis

The role of the lymph node stroma in cancer and aging

The function of the lymph node (LN) stroma is crucial for the induction of an adaptive immune response. The stroma includes lymphatic vessels, high endothelial venules (HEVs) and mesenchymal cells (MCs). In this thesis, I have been investigating the role of the LN stroma in cancer and aging. In Paper I, we demonstrate that LNs draining invasive breast cancer present remodeling of the HEVs and dysregulation of the perivascular MCs. Dilation of HEVs is correlating with inhibition of normal lymphocyte recruitment due to perivascular changes in the expression of molecular cues necessary for chemotaxis. As a follow up of Paper I, we created an automatic method for image analysis of HEVs by using artificial intelligence and deep learning. In paper II, we validate the HEV-finder that we suggest is a good automatic image analysis tool to study HEV remodeling in different subtypes of cancer and to perform survival studies in larger patient cohorts. In paper III, we present a mapping of the lymphatic endothelial cells (LECs) of the mouse and human LNs by using single-cell RNA sequencing and present a new LEC subset called PTX3-LECs. We demonstrate that both in mouse and human LNs there are four LEC subsets, forming the subcapsular and medullary sinuses where immune cells enter and exit the LN, respectively. Our mapping is a great tool for further investigation of different subsets and their specific genes in translational studies of homeostasis or disease. In paper IV, we are focusing on LN lipomatosis, which is an age-related phenomenon where the normal LN parenchyma is replaced by adipose tissue. Our data support that LN lipomatosis is developing in the medulla of the LN due to transdifferentiation of medullary MCs into adipocytes. We found that it is causing remodeling of the HEVs and loss of medullary lymphatic vessels, affecting both the entry and exit of lymphocytes. Based on our findings, we suggest that LN lipomatosis is a contributing factor in immunodeficiency in elderly. In paper V, we created a new transplantable breast cancer model in mouse to study the effect of LN metastasis by inducing the expression of the chemokine receptor CCR7 in the mammary carcinoma cell line EO771. The new model will allow us to study the effect of LN metastasis on the anti-tumor immunity and response to therapy. In summary, my thesis shed light on the importance of a functioning LN stroma in both cancer and aging and provides new tools for image analysis, translational studies of the LN stroma and models for LN metastasis

Lessons of Vascular Specialization From Secondary Lymphoid Organ Lymphatic Endothelial Cells

Secondary lymphoid organs, such as lymph nodes, harbor highly specialized and compartmentalized niches. These niches are optimized to facilitate the encounter of naive lymphocytes with antigens and antigen-presenting cells, enabling optimal generation of adaptive immune responses. Lymphatic vessels of lymphoid organs are uniquely specialized to perform a staggering variety of tasks. These include antigen presentation, directing the trafficking of immune cells but also modulating immune cell activation and providing factors for their survival. Recent studies have provided insights into the molecular basis of such specialization, opening avenues for better understanding the mechanisms of immune-vascular interactions and their applications. Such knowledge is essential for designing better treatments for human diseases given the central role of the immune system in infection, aging, tissue regeneration and repair. In addition, principles established in studies of lymphoid organ lymphatic vessel functions and organization may be applied to guide our understanding of specialization of vascular beds in other organs.De två första författarna delar förstaförfattarskapetDe två sista författarna delar sistaförfattarskapet</p

Low RAB6C expression is a predictor of tamoxifen benefit in estrogen receptor-positive/progesterone receptor-negative breast cancer

Over the last few decades, improved and more individualized treatment has contributed to the increased survival rate of patients with breast cancer. However, certain patients may receive excessive treatment resulting in undesired side effects. In a previous study, it was demonstrated that systemically untreated patients with estrogen receptor (ER)-positive/progesterone receptor (PR)-negative tumors with high Ras-related protein Rab-6C (RAB6C) expression levels (RAB6C(+)) had prolonged distant recurrence-free survival compared with that of patients exhibiting low RAB6C (RAB6C(-))-expressing tumors. The aim of the present study was to investigate whether RAB6C predicts the effectiveness of tamoxifen treatment. The present study used a dataset comprising 486 female patients with ER+ tumors from a randomized study conducted by the Stockholm Breast Cancer Study Group between November 1976 and August 1990. The patients were considered as low-risk if their tumor size was &amp;lt;= 30 mm and their lymph node status was negative. Patients were followed up until distant recurrence, mortality or when 25 years after randomization was achieved, whichever occurred first. For patients with ER+/PR-/RAB6C(+) tumors, prolonged distant recurrence-free survival could not be observed if the patients were treated with tamoxifen [hazard ratio (HR), 1.82; 95% confidence interval (CI), 0.69-4.79; P=0.23], whereas patients with ER+/PR-/RAB6C(-) tumors had 75% reduced distant recurrence risk (HR, 0.25; 95% CI, 0.09-0.70; P=0.008). In the ER+/PR+ subgroup, patients with RAB6C(-) and RAB6C(+) tumors benefited from tamoxifen treatment, though it was most evident in the RAB6C(+) group (HR, 0.27; 95% CI, 0.13-0.58; P=0.001). The results of the present study indicated that, for patients with ER+/PR- tumors, those with low RAB6C expression benefited from tamoxifen treatment, whereas no benefit was observed in patients with high RAB6C levels.Funding Agencies|Swedish Cancer SocietySwedish Cancer Society [17-0479]; Cancer Research Foundation of Radiumhemmet [181093]; King Gustav V Jubilee Clinical Research Foundation [181093]; Onkologiska klinikerna i Linkopings forskningsfond [2016-06-21]; ALF grants Region Ostergotland [LIO-795201]; County Council of Ostergotland [LIO-625491]; Cancer Society in Stockholm [181093]</p

Stromal transdifferentiation drives lipomatosis and induces extensive vascular remodeling in the aging human lymph node

Lymph node (LN) lipomatosis is a common, but rarely discussed phenomenon, associated with aging, involving a gradual exchange of the LN parenchyma into adipose tissue. The mechanisms behind these changes and the effects on the LN are unknown. We show that LN lipomatosis starts in the medullary regions of the human LN and link the initiation of lipomatosis to transdifferentiation of LN fibroblasts into adipocytes. The latter is associated with a downregulation of lymphotoxin beta expression. We also show that, isolated medullary and CD34+ fibroblast, in contrast to the reticular cells of the T-cell zone, display an inherent higher sensitivity for adipogenesis. Progression of lipomatosis leads to a gradual loss of the medullary lymphatic network, but at later stages, collecting-like lymphatic vessels, are found inside the adipose tissue. The stromal dysregulation includes a dramatic remodeling and dilation of the high endothelial venules associated with reduced density of naïve T-cells. Abnormal clustering of plasma cells is also observed. Thus, LN lipomatosis causes widespread stromal dysfunction with consequences for the immune contexture of the human LN. Our data warrant an increased awareness of LN lipomatosis as a factor contributing to decreased immune functions in the elderly and in disease

RAB6C is an independent prognostic factor of estrogen receptor-positive/progesterone receptor-negative breast cancer

The majority of breast cancer tumors are estrogen receptor-positive (ER+) and can be treated with endocrine therapy. However, certain patients may exhibit a good prognosis without systemic treatment. The aim of the present study was to identify novel prognostic factors for patients with ER breast cancer tumors using gene copy data, and to investigate if these factors have prognostic value in subgroups categorized by progesterone receptor status (PR). Public data, including the whole genome gene copy data of 199 systemically untreated patients with ER+ tumors, were utilized in the present study. To assess prognostic value, patients were divided into two groups using the median gene copy number as a cut-off for the SNPs that were the most variable. One SNP was identified, which indicated that the Ras-related protein Rab-6C (RAB6C) gene may exhibit prognostic significance. Therefore, RAB6C protein expression was subsequently investigated in a second independent cohort, consisting of 469 systematically untreated patients (of which 310 were ER+) who received long term follow-up. In the public data set, a distant recurrence risk reduction of 55% was determined for copy numbers above the median value of RAB6C compared with numbers below [multivariable adjusted hazard ratio (HR), 0.45; 95% CI 0.28-0.72; P=0.001)]. It was also more pronounced in the ER+/PR- subgroup (HR, 0.15; 95% CI, 0.05-0.46; P=0.001). In the second cohort, patients of the ER+/PR- subgroup who exhibited high RAB6C expression had a reduced distant recurrence risk (HR, 0.17; 95% CI, 0.05-0.60; P=0.006). However, this was not identified among ER+/PR- tumors (HR, 1.31; 95% CI, 0.69-2.48; P=0.41). The results of the present study indicated that RAB6C serves as an independent prognostic factor of distant recurrence risk in systemically untreated patients with an ER+/PR- tumor.Funding Agencies|Swedish Cancer SocietySwedish Cancer Society [17-0479]; Cancer Research Foundations of Radiumhemmet; Cancer Society in Stockholm; King Gustav V Jubilee Clinical Research Foundation [181093]; Onkologiska klinikerna i Linkopings forskningsfond [2016-06-21]; ALF grants Region Ostergotland [LIO-795201]; County Council of Ostergotland [LIO-625491]</p

Remodeling of the Lymph Node High Endothelial Venules Reflects Tumor Invasiveness in Breast Cancer and is Associated with Dysregulation of Perivascular Stromal Cells

The tumor-draining lymph nodes (TDLNs) are primary sites for induction of tumor immunity. They are also common sites of metastasis, suggesting that tumor-induced mechanisms can subvert anti-tumor immune responses and promote metastatic seeding. The high endothelial venules (HEVs) together with CCL21-expressing fibroblastic reticular cells (FRCs) are essential for lymphocyte recruitment into the LNs. We established multicolor antibody panels for evaluation of HEVs and FRCs in TDLNs from breast cancer (BC) patients. Our data show that patients with invasive BC display extensive structural and molecular remodeling of the HEVs, including vessel dilation, thinning of the endothelium and discontinuous expression of the HEV-marker PNAd. Remodeling of the HEVs was associated with dysregulation of CCL21 in perivascular FRCs and with accumulation of CCL21-saturated lymphocytes, which we link to loss of CCL21-binding heparan sulfate in FRCs. These changes were rare or absent in LNs from patients with non-invasive BC and cancer-free organ donors and were observed independent of nodal metastasis. Thus, pre-metastatic dysregulation of core stromal and vascular functions within TDLNs reflect the primary tumor invasiveness in BC. This adds to the understanding of cancer-induced perturbation of the immune response and opens for prospects of vascular and stromal changes in TDLNs as potential biomarkers. Simple Summary Tumor draining lymph nodes (TDLNs) are the most common metastatic sites in human cancer but are also essential sites for induction of tumor immunity. How different types of primary tumors affect the anti-tumor immune response in the LNs is not fully understood. By analyzing biobank tissue from breast cancer patients, we demonstrate that invasive breast cancer induce dramatic pre-metastatic LN changes affecting the structure and function of the specialized LN vasculature and associated stromal cells, required for recruitment of T-lymphocytes into the LNs. These changes could not be seen in patients with non-invasive breast cancer and provide new insights of how invasive tumors can disrupt essential functions within the immune system. The data also shows promise of LN stromal and vascular changes as possible future biomarkers for prediction of disease progression in human cancer

Automated detection of vascular remodeling in human tumor draining lymph nodes by the deep learning tool HEV-finder

Vascular remodeling is common in human cancer and has potential as future biomarkers for prediction of disease progression and tumor immunity status. It can also affect metastatic sites, including the tumor-draining lymph nodes (TDLNs). Dilation of the high endothelial venules (HEVs) within TDLNs has been observed in several types of cancer. We recently demonstrated that it is a premetastatic effect that can be linked to tumor invasiveness in breast cancer. Manual visual assessment of changes in vascular morphology is a tedious and difficult task, limiting high-throughput analysis. Here we present a fully automated approach for detection and classification of HEV dilation. By using 12,524 manually classified HEVs, we trained a deep-learning model and created a graphical user interface for visualization of the results. The tool, named the HEV-finder, selectively analyses HEV dilation in specific regions of the lymph nodes. We evaluated the HEV-finder's ability to detect and classify HEV dilation in different types of breast cancer compared to manual annotations. Our results constitute a successful example of large-scale, fully automated, and user-independent, image-based quantitative assessment of vascular remodeling in human pathology and lay the ground for future exploration of HEV dilation in TDLNs as a biomarker