Fibroblast heterogeneity and functions: insights from single-cell sequencing in wound healing, breast cancer, ovarian cancer and melanoma.

Cancer has been described as the wound that does not heal, in large part due to fibroblast involvement. Activation of cancer-associated fibroblasts (CAFs) contributes to critical features of the tumor microenvironment, including upregulation of key marker proteins, recruitment of immune cells, and deposition of extracellular matrix (ECM)-similar to fibroblast activation in injury-induced wound healing. Prior to the widespread availability of single-cell RNA sequencing (scRNA seq), studies of CAFs or fibroblasts in wound healing largely relied on models guided by individual fibroblast markers, or methods with less resolution to unravel the heterogeneous nature of CAFs and wound healing fibroblasts (especially regarding scarring outcome). Here, insights from the enhanced resolution provided by scRNA sequencing of fibroblasts in normal wound healing, breast cancer, ovarian cancer, and melanoma are discussed. These data have revealed differences in expression of established canonical activation marker genes, epigenetic modifications, fibroblast lineages, new gene and proteins of clinical interest for further experimentation, and novel signaling interactions with other cell types that include spatial information.

Autophagy in stromal fibroblasts promotes tumor desmoplasia and mammary tumorigenesis.

Autophagy inhibitors are currently being evaluated in clinical trials for the treatment of diverse cancers, largely due to their ability to impede tumor cell survival and metabolic adaptation. More recently, there is growing interest in whether and how modulating autophagy in the host stroma influences tumorigenesis. Fibroblasts play prominent roles in cancer initiation and progression, including depositing type 1 collagen and other extracellular matrix (ECM) components, thereby stiffening the surrounding tissue to enhance tumor cell proliferation and survival, as well as secreting cytokines that modulate angiogenesis and the immune microenvironment. This constellation of phenotypes, pathologically termed desmoplasia, heralds poor prognosis and reduces patient survival. Using mouse mammary cancer models and syngeneic transplantation assays, we demonstrate that genetic ablation of stromal fibroblast autophagy significantly impedes fundamental elements of the stromal desmoplastic response, including collagen and proinflammatory cytokine secretion, extracellular matrix stiffening, and neoangiogenesis. As a result, autophagy in stromal fibroblasts is required for mammary tumor growth in vivo, even when the cancer cells themselves remain autophagy-competent . We propose the efficacy of autophagy inhibition is shaped by this ability of host stromal fibroblast autophagy to support tumor desmoplasia.

The LC3-conjugation machinery specifies the loading of RNA-binding proteins into extracellular vesicles.

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.

Unraveling the Breast: Advances in Mammary Biology and Cancer Methods.

The field of mammary gland biology and breast cancer research encompasses a wide range of topics and scientific questions, which span domains of molecular, cell and developmental biology, cancer research, and veterinary and human medicine, with interdisciplinary overlaps to non-biological domains. Accordingly, mammary gland and breast cancer researchers employ a wide range of molecular biology methods, in vitro techniques, in vivo approaches as well as in silico analyses. The list of techniques is ever-expanding; together with the refinement of established, staple techniques in the field, new technologies keep emerging thanks to technological advances and scientific creativity. This issue of the Journal of Mammary Gland Biology and Neoplasia represents a compilation of original articles and reviews focused on methods used in mammary gland biology and breast cancer research.

S100a4-Cre-mediated deletion of Patched1 causes hypogonadotropic hypogonadism: role of pituitary hematopoietic cells in endocrine regulation.

Hormones produced by the anterior pituitary gland regulate an array of important physiological functions, but pituitary hormone disorders are not fully understood. Herein we report that genetically-engineered mice with deletion of the hedgehog signaling receptor Patched1 by S100a4 promoter-driven Cre recombinase (S100a4-Cre;Ptch1fl/fl mutants) exhibit adult-onset hypogonadotropic hypogonadism and multiple pituitary hormone disorders. During the transition from puberty to adult, S100a4-Cre;Ptch1fl/fl mice of both sexes develop hypogonadism coupled with reduced gonadotropin levels. Their pituitary glands also display severe structural and functional abnormalities, as revealed by transmission electron microscopy and expression of key genes regulating pituitary endocrine functions. S100a4-Cre activity in the anterior pituitary gland is restricted to CD45+ cells of hematopoietic origin, including folliculo-stellate cells and other immune cell types, causing sex-specific changes in the expression of genes regulating the local microenvironment of the anterior pituitary. These findings provide in vivo evidence for the importance of pituitary hematopoietic cells in regulating fertility and endocrine function, in particular during sexual maturation and likely through sexually dimorphic mechanisms. These findings support a previously unrecognized role of hematopoietic cells in causing hypogonadotropic hypogonadism and provide inroads into the molecular and cellular basis for pituitary hormone disorders in humans.

Inflammatory signaling cascades and autophagy in cancer.

Tumor-associated inflammation is predictive of poor prognosis and drives a variety of tumorigenic phenotypes, including tumor proliferation and survival, angiogenesis, invasiveness, and metastasis. Here, we review mammalian data addressing the interaction of macroautophagy/autophagy with key signaling cascades associated with tumor inflammation. Although our understanding of this area remains incomplete, certain inflammatory pathways have emerged as important mediators of the crosstalk between autophagy and inflammation in tumors. Consistent with the multifaceted roles for autophagy in tumor cells, results to date support the hypothesis that inflammatory pathways can suppress or induce autophagy in a context-dependent manner; in turn, autophagy suppresses or promotes inflammation in cancers. Furthermore, emerging data suggest that autophagy may influence cytokine production and secretion via diverse mechanisms, which has implications for the immune and inflammatory microenvironment in tumors.

New paradigms for the Hedgehog signaling network in mammary gland development and breast Cancer.

The Hedgehog signaling network regulates organogenesis, cell fate, proliferation, survival, and stem cell self-renewal in many mammalian tissues. Aberrant activation of the Hedgehog signaling network is present in ~25% of all cancers, including breast. Altered expression of Hedgehog network genes in the mammary gland can elicit phenotypes at many stages of development. However, synthesizing a cohesive mechanistic model of signaling at different stages of development has been difficult. Emerging data suggest that this difficulty is due, in part, to non-canonical and tissue compartment-specific (i.e., epithelial, versus stromal, versus systemic) functions of Hedgehog network components. With respect to systemic functions, Hedgehog network genes regulate development of endocrine organs that impinge on mammary gland development extrinsically. These new observations offer insight into previously conflicting data, and have bearing on the potential for anti-Hedgehog therapeutics in the treatment of breast cancer.

Epithelial and non-epithelial Ptch1 play opposing roles to regulate proliferation and morphogenesis of the mouse mammary gland.

Patched 1 (Ptch1) has epithelial, stromal and systemic roles in murine mammary gland organogenesis, yet specific functions remain undefined. Cre-recombinase-mediated Ptch1 ablation in mammary epithelium increased proliferation and branching, but did not phenocopy transgenic expression of activated smoothened (SmoM2). The epithelium showed no evidence of canonical hedgehog signaling, and hyperproliferation was not blocked by smoothened (SMO) inhibition, suggesting a non-canonical function of PTCH1. Consistent with this possibility, nuclear localization of cyclin B1 was increased. In non-epithelial cells, heterozygous Fsp-Cre-mediated Ptch1 ablation increased proliferation and branching, with dysplastic terminal end buds (TEB) and ducts. By contrast, homozygous Ptch1 ablation decreased proliferation and branching, producing stunted ducts filled with luminal cells showing altered ovarian hormone receptor expression. Whole-gland transplantation into wild-type hosts or estrogen/progesterone treatment rescued outgrowth and hormone receptor expression, but not the histological changes. Bone marrow transplantation failed to rescue outgrowth. Ducts of Fsp-Cre;Ptch1fl/fl mice were similar to Fsp-Cre;SmoM2 ducts, but Fsp-Cre;SmoM2 outgrowths were not stunted, suggesting that the histology might be mediated by Smo in the local stroma, with systemic Ptch1 required for ductal outgrowth and proper hormone receptor expression in the mammary epithelium.

Urinary Peptide patterns in native kidneys and kidney allografts.

BACKGROUND: The use of biopsies to determine kidney health after kidney transplantation is an invasive procedure with some risk to the patient. Consequently, a noninvasive test for transplanted kidney health would provide a significant advantage over current clinical practice. METHODS: Urines from kidney donors before nephrectomy, pretransplant patients with native kidney disease, and posttransplant kidney recipients were examined for protein biomarkers to diagnose or prognose kidney disease. Proteins were extracted by C4 reverse phase affinity and analyzed by matrix assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS: Urine from individuals with healthy kidneys showed few components other than two ubiquitous saposin B glycoisoforms. Patients with kidney disease lacked saposin B and showed new components in two patterns: the most common contained beta-2 microglobulin (B2M, m/z=11,732) plus one or more peaks at m/z=10,350, 9480, 4337, and 4180. Pattern 2 lacked beta-2 microglobulin but contained several degradation products of alpha-1 antitrypsin. Other pathologic components included urinary protein 1 (m/z=15,835), transthyretin (m/z=13,880), and a component at m/z=13,350. CONCLUSIONS: Patients with acute rejection showed profiles that ranged from those of kidney donors to those of advanced kidney disease. The range of patterns may be useful for analysis of transplant patients without complications and persons with developing kidney disease before or after transplant.

O-glycoside biomarker of apolipoprotein C3: responsiveness to obesity, bariatric surgery, and therapy with metformin, to chronic or severe liver disease and to mortality in severe sepsis and graft vs host disease.

The glyco-isoforms of intact apolipoprotein C3 (ApoC3) were used to probe glycomic changes associated with obesity and recovery following bariatric surgery, liver diseases such as chronic hepatitis C (CHC) and alcoholic liver cirrhosis, as well as severe, multiorgan diseases such as sepsis and graft vs host disease (GVHD). ApoC3 glyco-isoform ratios responded to unique stimuli that did not correlate with serum lipids or with other blood components measured in either a control population or a group of extremely obese individuals. However, glyco-isoform ratios correlated with obesity with a 1.8-fold change among subjects eligible for bariatric surgery relative to a nonobese control population. Bariatric surgery resulted in rapid change of isoform distribution to that of nonobese individuals, after which the distribution was stable in each individual. Although multiple simultaneous factors complicated effector attribution, the isoform ratios of very obese individuals were nearly normal for diabetic individuals on metformin therapy. Glyco-isoform ratios were sensitive to liver diseases such as chronic hepatitis C and alcoholic liver cirrhosis. The correlation coefficient with fibrosis was superior to that of current assays of serum enzyme levels. Diseases of pregnancy that can result in liver damage, HELLP syndrome and pre-eclampsia, did not alter ApoC3 glyco-isoform ratios. Early after umbilical cord blood transplantation the isoform ratios changed and returned to normal in long-term survivors. Larger changes were observed in persons who died. GVHD had little effect. Persons with severe sepsis showed altered ratios. Similar cut-points for mortality (3.5-fold difference from controls) were found for UCBT and sepsis. Similar values characterized liver cirrhosis. Overall, while changes of glyco-isoform ratios occurred in many situations, individual stability of isoform distribution was evident and large changes were limited to high-level disease. If ratio changes associated with obesity are found to document a risk factor for long-term outcomes, the information provided by glyco-isoform ratio changes may provide important, novel information for diagnostic, prognostic and therapy response to metabolic conditions.