Tendon is a dense connective tissue that stores and transmits forces between muscles and bones. Cellular heterogeneity is increasingly recognized as an important factor in the biological basis of tissue homeostasis and disease, yet little is known about the diversity of cell types that populate tendon. To address this, we determined the heterogeneity of cell populations within mouse Achilles tendons using single-cell RNA sequencing. In assembling a transcriptomic atlas of Achilles tendons, we identified 11 distinct types of cells, including 3 previously undescribed populations of tendon fibroblasts. Prior studies have indicated that pericytes, which are found in the vasculature of tendons, could serve as a potential source of progenitor cells for adult tendon fibroblasts. Using trajectory inference analysis, we provide additional support for the notion that pericytes are likely to be at least one of the progenitor cell populations for the fibroblasts that compose adult tendons. We also modeled cell-cell interactions and identified previously undescribed ligand-receptor signaling interactions involved in tendon homeostasis. Our novel and interactive tendon atlas highlights previously underappreciated heterogeneity between and within tendon cell populations. The atlas also serves as a resource to further the understanding of tendon extracellular matrix assembly, maintenance, and in the design of therapies for tendinopathies.

Bone metastasis is an incurable complication of breast cancer affecting 70-80 % of advanced patients. It is a multistep process that includes tumour cell mobilisation, intravasation, survival in the circulation, extravasation, migration and proliferation in the bone marrow/bone. Although novel findings demonstrate the bone marrow microenvironment significance in bone metastatic progression, a majority of studies have focused on end-stage disease and little is known about how the pre-metastatic niche arises in the bone marrow/bone tissues. We demonstrated a significant increase in patients' peripheral blood plasma ability to induce transendothelial migration of MCF-7 cells compared with healthy volunteers. Moreover, high RANKL, MIF and OPG levels in patients' peripheral blood could play a role in the intravasation, angiogenesis, survival and epithelial-mesenchymal transition of circulating tumour cells. Also, we observed a significant increase in patients' bone marrow plasma capacity to induce transendothelial migration of MDA-MB231 and MCF-7 cells compared with healthy volunteers. Furthermore, patients' bone marrow mesenchymal stem cells could control the recruitment of tumour cells, modifying the MCF-7 and MDA-MB231 cell migration. In addition, we found a significantly higher MDA-MB231 cell proliferation when we used patients' bone marrow plasma compared with healthy volunteers. Interestingly, PDGF-AB, ICAM-1 and VCAM-1 levels in patients' bone marrow were significantly higher than the values of healthy volunteers, suggesting that they could be involved in the cancer cell extravasation, bone resorption and cancer cell proliferation. We believe that these results can reveal new information about what alterations happen in the bone marrow of advanced breast cancer patients before bone colonisation, changes that create optimal soil for the metastatic cascade progression.

Ductal carcinoma in situ (DCIS) is a non-obligate precursor of breast cancer, and it only progresses to invasive breast cancer in around 40% of patients. While immune infiltrates have been observed in these early cancer lesions, their potential prognostic value is still unclear. Regulatory T (Treg) cells accumulate in advanced breast cancers, and predict poor outcome. We have shown before that ablation of Treg cells in established tumors leads to significant decrease in primary and metastatic tumor burden. In this work, we sought to investigate Treg cell function in the progression from non-invasive to invasive breast cancer lesions. To this end, we used the murine mammary tumor virus polyoma middle T (MMTV-PyMT) murine model of spontaneous, stage-wise breast carcinogenesis crossed to Foxp3 DTR knock in mice, allowing Treg cell ablation by administration of diphtheria toxin. Transient targeting of Treg cells at the in situ carcinoma stage resulted in a significant increase in the number of tumor-bearing mammary glands and size of growing tumors compared with control mice. Whole mammary gland mounts and histological examination confirmed larger emergent tumor area in Treg cell-ablated mice, and revealed that these tumors were characterized by a more advanced tumor staging, with presence of early invasion, increased desmoplasia and collagen deposition. Furthermore, Treg cell ablation increased the percentage of cancer stem/progenitor cells in the mammary compartment. Interestingly, Treg cell ablation resulted in increased inflammatory cytokines IL-4 and IL-5 with a concomitant reduction in classically activated tumor associated macrophages. This TH2-biased immune regulatory mammary inflammation was consistent with the enhancement in tumor promotion that we observed. Overall, our study demonstrates that Treg cells oppose breast cancer progression at early stages, raising a cautionary note regarding the consideration of immune intervention targeted at boosting immune responses for DCIS.

Aims Breast cancer is the most frequently diagnosed and leading cause of cancer death among women worldwide. It was classified within molecular intrinsic subtypes: luminal A, luminal B, human epidermal growth factor receptor 2‐enriched and basal‐like. Epinephrine and norepinephrine, released during stress, bind to adrenoceptors. α2‐adrenoceptors are encoded by the ADRA2A, ADRA2B and ADRA2C genes and β2 by ADRB2. Methods We compiled several publicly available Affymetrix gene expression datasets, obtaining a large cohort of 1924 patients with distant metastasis‐free survival (DMFS) data and evaluated the association between adrenoceptor expression, clinicopathological markers and outcome. Results ADRA2A high expressing tumours also expressed hormone receptors and presented diminished tumour size, grade and not compromised lymph nodes. ADRB2 high expression was found in smaller, low grade, oestrogen receptor‐positive tumours. Both were significantly associated with the absence of metastasis. High expression of ADRA2C was positively associated with increased tumour size and metastatic relapse. We observed a significant increase in DMFS of patients with high ADRA2A (hazard ratio 0.54, 95% CI 0.45–0.65, P < .001) and ADRB2 (0.77, 0.64–0.93, P = .006) expression and a decrease with ADRA2C high expression (1.45, 1.16–1.81, P = .001). For patients with luminal tumours, ADRA2A was the only factor that retained its significance as an independent predictor of DMFS while ADRA2C expression was an independent predictor for worse prognosis in basal‐like tumours. Conclusions We herein provide new insight for a potential role of ADRA2A and ADRA2C in breast cancer. In low‐ and medium‐income countries, their incorporation to routine immunohistochemistry analysis of biopsies or tumour samples, could provide additional low‐cost prognostic factors.

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Several studies have confirmed that the breast tumor microenvironment drives cancer progression and metastatic development. The aim of our research was to investigate the prognostic significance of the breast tumor microenvironment in untreated early breast cancer patients. Therefore, we analyzed the association of the expression of α-SMA, FSP, CD105 and CD146 in CD34-negative spindle-shaped stromal cells, not associated with the vasculature, in primary breast tumors with classical prognostic marker levels, metastatic recurrence, local relapse, disease-free survival, metastasis-free survival and the overall survival of patients. In the same way, we evaluated the association of the amount of intra-tumor stroma, fibroblasts, collagen deposition, lymphocytic infiltration and myxoid changes in these samples with the clinical-pathological data previously described. This study is the first to demonstrate the high CD105 expression in this stromal cell type as a possible independent marker of unfavorable prognosis in early breast cancer patients. Our study suggests that this new finding can be useful prognostic marker in the clinical-pathological routine.

Tendon injuries are a common clinical condition with limited treatment options. The cellular components of the innate immune system, such as neutrophils and macrophages, have been studied in tendon injuries. However, the adaptive immune system, comprising specialized lymphocytes, plays an important role in orchestrating the healing of numerous tissues, but less is known about these cells in tendon healing. To gain a greater understanding of the biological processes that regulate tendon healing, we determined how the cellular components of the adaptive and innate immune system respond to a tendon injury using two-month-old male mice. We observed that lymphatic vasculature is present in the epitenon and superficial regions of Achilles tendons, and that the lymphatics drain into the popliteal lymph node. We then created an acute Achilles tenotomy followed by repair, and collected tendons and popliteal lymph nodes 1, 2, and 4 wk after injury. Tendon injury resulted in a robust adaptive immune cell response that followed an initial innate immune cell response in tendons and lymph nodes. Monocytes, neutrophils, and macrophages initially accumulated at 1 wk after injury in tendons, while dendritic cells and CD4+ T cells peaked at 2 wk after injury. B cells and CD8+ T cells progressively increased over time. In parallel, immune cells of the popliteal lymph node demonstrated a similarly coordinated response to the injury. These results suggest that there is an adaptive immune response to tendon injury, and adaptive immune cells may play a role in regulating tendon healing. NEW & NOTEWORTHY While the innate immune system, consisting of macrophages and related hematopoietic cells, has been studied in tendon injury, less is known about the adaptive immune system. Using a mouse model of Achilles tendon tenotomy and repair, we observed an adaptive immune cell response, consisting of CD4+ and CD8+ T cells, and B cells, which occur through 4 wk after tendon injury. This response appeared to be coordinated by the draining popliteal lymph node.