The mesenchymal stromal cell secretome promotes tissue regeneration and increases macrophage infiltration in acute and methicillin-resistant Staphylococcus aureus-infected skin wounds in vivo.

BACKGROUND AIMS: The prevalence of chronic wounds continues to be a burden in human medicine. Methicillin-resistant Staphylococcus aureus (MRSA) is commonly isolated from infected wounds. MRSA infections primarily delay healing by impairing local immune cell functions. This study aimed to investigate the potential of mesenchymal stromal cell (MSC)-secreted bioactive factors, defined as the secretome, to improve innate immune responses in vivo. MSCs were isolated from the bone marrow of horses, which serve as valuable translational models for wound healing. The MSC secretome, collected as conditioned medium (CM), was evaluated in vivo using mouse models of acute and MRSA-infected skin wounds. METHODS: Punch biopsies were used to create two full-thickness skin wounds on the back of each mouse. Acute wounds were treated daily with control medium or bone marrow-derived MSC (BM-MSC) CM. The antibiotic mupirocin was administered as a positive control for the MRSA-infected wound experiments. Wounds were photographed daily, and wound images were measured to determine the rate of closure. Trichrome staining was carried out to examine wound tissue histologically, and immunofluorescence antibody binding was used to assess immune cell infiltration. Wounds in the MRSA-infected model were swabbed for quantification of bacterial load. RESULTS: Acute wounds treated with BM-MSC CM showed accelerated wound closure compared with controls, as illustrated by enhanced granulation tissue formation and resolution, increased vasculature and regeneration of hair follicles. This treatment also led to increased neutrophil and macrophage infiltration. Chronic MRSA-infected wounds treated with BM-MSC CM showed reduced bacterial load accompanied by better resolution of granulation tissue formation and increased infiltration of pro-healing M2 macrophages compared with control-treated infected wounds. CONCLUSIONS: Collectively, our findings indicate that BM-MSC CM exerts pro-healing, immunomodulatory and anti-bacterial effects on wound healing in vivo, validating further exploration of the MSC secretome as a novel treatment option to improve healing of both acute and chronic wounds, especially those infected with antibiotic-resistant bacteria.

Characterization of HphA: the First Enzyme in the Homologation Pathway of l-Phenylalanine and l-Tyrosine.

Homologation of amino acids is the insertion or deletion of a methylene group to their side chain, which is a relatively uncommon chemical transformation observed in peptide natural product (NP) structure. Homologated amino acids can potentially make the NP more stable in a biological system, but its biosynthesis is yet to be understood. This study biochemically characterized the first of three unexplored enzymes in the homologation pathway of l-phenylalanine and l-tyrosine. Previously proposed reactions catalyzed by HphA were confirmed by reversed-phase high-performance liquid chromatography and tandem mass spectrometry analysis. The substrate profile and kinetic parameters showed high selectivity for the natural substrates and their close analogs. The comparability of HphA to homologous enzymes in primary metabolic pathways, 2-isopropylmate synthase and homocitrate synthase which are involved in l-leucine and l-lysine biosynthesis, respectively, was validated by bioinformatical and site-directed mutagenesis studies. The knowledge obtained from this study has deepened the understanding of the homologation of amino acids, which can lead to future combinatorial biosynthesis and metabolic engineering studies.

Effect of pregnancy on isolation efficiency and in vitro proliferation of equine peripheral-blood derived mesenchymal stromal cells.

Mesenchymal stromal cells (MSCs) have regenerative and immunomodulatory potential and may be used to treat injured tissues. Pregnancy has been associated with increased MSCs in the peripheral circulation in multiple species, but to date, there are no reports on this matter in horses. This study aimed to evaluate the effect of pregnancy on isolation efficiency and proliferation capacity of equine MSCs derived from the peripheral blood (PB) of mares. Venous blood samples were collected at the 11th month of gestation and 1 month after delivery from clinically healthy Arabian mares that presented normal pregnancies. Blood samples were processed for in vitro cellular culture and hormonal and metabolic profiles. MSCs were isolated and characterized by trilineage differentiation potential, immunophenotyping, analyzed by gene sequencing and proliferation assays. The isolation of peripheral blood mononuclear cells (PBMCs) of pregnant mares were associated with higher isolation efficiency and proliferative capacity of MSCs derived from peripheral blood (PB-MSCs) recovered pre-partum than those isolated post-partum. Although fetal gender, parity, 5α-reduced pregnanes, insulin, and cortisol were shown to affect cellular proliferation, individual factors and the small population studied must be considered. This study suggests that PB-MSCs from pregnant mares could be a valuable alternative source of MSCs for therapeutic purposes.

Natural Products That Contain Higher Homologated Amino Acids.

This review focuses on discussing natural products (NPs) that contain higher homologated amino acids (homoAAs) in the structure as well as the proposed and characterized biosynthesis of these non-proteinogenic amino acids. Homologation of amino acids includes the insertion of a methylene group into its side chain. It is not a very common modification found in NP biosynthesis as approximately 450 homoAA-containing NPs have been isolated from four bacterial phyla (Cyanobacteria, Actinomycetota, Myxococcota, and Pseudomonadota), two fungal phyla (Ascomycota and Basidiomycota), and one animal phylum (Porifera), except for a few examples. Amino acids that are found to be homologated and incorporated in the NP structures include the following ten amino acids: alanine, arginine, cysteine, isoleucine, glutamic acid, leucine, phenylalanine, proline, serine, and tyrosine, where isoleucine, leucine, phenylalanine, and tyrosine share the comparable enzymatic pathway. Other amino acids have their individual homologation pathway (arginine, proline, and glutamic acid for bacteria), likely utilize the primary metabolic pathway (alanine and glutamic acid for fungi), or have not been reported (cysteine and serine). Despite its possible high potential in the drug discovery field, the biosynthesis of homologated amino acids has a large room to explore for future combinatorial biosynthesis and metabolic engineering purpose.

A xenotransplantation mouse model to study physiology of the mammary gland from large mammals.

Although highly conserved in structure and function, many (patho)physiological processes of the mammary gland vary drastically between mammals, with mechanisms regulating these differences not well understood. Large mammals display variable lactation strategies and mammary cancer incidence, however, research into these variations is often limited to in vitro analysis due to logistical limitations. Validating a model with functional mammary xenografts from cryopreserved tissue fragments would allow for in vivo comparative analysis of mammary glands from large and/or rare mammals and would improve our understanding of postnatal development, lactation, and premalignancy across mammals. To this end, we generated functional mammary xenografts using mammary tissue fragments containing mammary stroma and parenchyma isolated via an antibody-independent approach from healthy, nulliparous equine and canine donor tissues to study these species in vivo. Cryopreserved mammary tissue fragments were xenotransplanted into de-epithelialized fat pads of immunodeficient mice and resulting xenografts were structurally and functionally assessed. Preimplantation of mammary stromal fibroblasts was performed to promote ductal morphogenesis. Xenografts recapitulated mammary lobule architecture and contained donor-derived stromal components. Mammatropic hormone stimulation resulted in (i) upregulation of lactation-associated genes, (ii) altered proliferation index, and (iii) morphological changes, indicating functionality. Preimplantation of mammary stromal fibroblasts did not promote ductal morphogenesis. This model presents the opportunity to study novel mechanisms regulating unique lactation strategies and mammary cancer induction in vivo. Due to the universal applicability of this approach, this model serves as proof-of-concept for developing mammary xenografts for in vivo analysis of virtually any mammals, including large and rare mammals.

miRNA-214-3p stimulates carcinogen-induced mammary epithelial cell apoptosis in mammary cancer-resistant species.

Mammary cancer incidence varies greatly across species and underlying mechanisms remain elusive. We previously showed that mammosphere-derived epithelial cells from species with low mammary cancer incidence, such as horses, respond to carcinogen 7, 12-Dimethylbenz(a)anthracene-induced DNA damage by undergoing apoptosis, a postulated anti-cancer mechanism. Additionally, we found that miR-214-3p expression in mammosphere-derived epithelial cells is lower in mammary cancer-resistant as compared to mammary cancer-susceptible species. Here we show that increasing miR-214 expression and decreasing expression of its target gene nuclear factor kappa B subunit 1 in mammosphere-derived epithelial cells from horses abolishes 7,12-Dimethylbenz(a)anthracene-induced apoptosis. A direct interaction of miR-214-3p with another target gene, unc-5 netrin receptor A, is also demonstrated. We propose that relatively low levels of miR-214 in mammosphere-derived epithelial cells from mammals with low mammary cancer incidence, allow for constitutive gene nuclear factor kappa B subunit 1 expression and apoptosis in response to 7, 12-Dimethylbenz(a)anthracene. Better understanding of the mechanisms regulating cellular responses to carcinogens improves our overall understanding of mammary cancer resistance mechanisms.

Bovine milk-derived cells express transcriptome markers of pluripotency and secrete bioactive factors with regenerative and antimicrobial activity.

The bovine mammary stem/progenitor cell secretome stimulates regeneration in vitro and contains proteins associated with antimicrobial defense. This has led to the exploration of the secretome as a biologic treatment for mastitis, a costly inflammation of the udder commonly caused by bacteria. This study reports on a population of bovine mammary stem/progenitor cells isolated non-invasively from milk (MiDCs). MiDCs were characterized by immunophenotyping, mammosphere formation assays, and single cell RNA sequencing. They displayed epithelial morphology, exhibited markers of mammary stem/progenitor cells, and formed mammospheres, like mammary gland tissue-isolated stem/progenitor cells. Single cell RNA sequencing revealed two sub-populations of MiDCs: epithelial cells and macrophages. Functionally, the MiDC secretome increased fibroblast migration, promoted angiogenesis of endothelial cells, and inhibited the growth of mastitis-associated bacteria, including antibiotic-resistant strains, in vitro. These qualities of MiDCs render them a source of stem cells and stem cell products that may be used to treat diseases affecting the dairy industry, including mastitis.

Use of Biologics and Stem Cells for Wound Healing in the Horse.

Treatment of skin wounds is a high priority in veterinary medicine because healthy uncompromised skin is essential for the well-being of horses. Stem cells and other biologic therapies offer benefits by reducing the need for surgical procedures and conventional antibiotics. Evidence from in vitro studies and small in vivo trials supports the use of equine stem cells and biologics for the treatment of acute and chronic cutaneous wounds. Larger clinical trials are warranted to better evaluate the regenerative and immunological responses to these treatments. Additionally, delivery methods and treatment schedules should be optimized to improve efficacy of these novel therapies.

Induced pluripotent stem cells from domesticated ruminants and their potential for enhancing livestock production.

Ruminant livestock, including cattle, sheep, goat, and buffalo, are essential for global food security and serve valuable roles in sustainable agricultural systems. With the limited availability of embryonic stem cells (ESCs) from these species, ruminant induced pluripotent stem cells (iPSCs) and iPSC-like cells provide a valuable research tool for agricultural, veterinary, biomedical, and pharmaceutical applications, as well as for the prospect of translation to human medicine. iPSCs are generated by reprogramming of adult or fetal cells to an ESC-like state by ectopic expression of defined transcription factors. Despite the slow pace the field has evolved in livestock species compared to mice and humans, significant progress has been made over the past 15 years in using different cell sources and reprogramming protocols to generate iPSCs/iPSC-like cells from ruminants. This mini review summarizes the current literature related to the derivation of iPSCs/iPSC-like cells from domesticated ruminants with a focus on reprogramming protocols, characterization, associated limitations, and potential applications in ruminant basic science research and production.

Mesenchymal stromal cells isolated from chicken peripheral blood secrete bioactive factors with antimicrobial and regenerative properties.

Mesenchymal stromal cells (MSCs) are adult multipotent progenitor cells that have been isolated from various tissue sources of many species, primarily mammals. Generally, these cells proliferate extensively in culture and have been shown to secrete bioactive factors that contribute to healing processes by regulating inflammation, modulating immune responses, inhibiting bacterial growth, and promoting tissue regeneration. The present study reports on the isolation and characterization of MSCs from the peripheral blood (PB) of chickens. Chicken PBMSCs were characterized based on their trilineage differentiation potential and gene and protein expression of MSC-specific cell surface markers. To determine functionality, conditioned medium (CM), which contains all bioactive factors secreted by MSCs, was collected from chicken PBMSCs, and used in in vitro antimicrobial, migration, and angiogenesis assays. Chicken PBMSC CM was found to (i) inhibit the growth of planktonic Staphylococcus aureus (S. aureus), and even more significantly the methicillin-resistant S. aureus (MRSA), (ii) decrease adhesion and promote migration of fibroblasts, and (iii) support endothelial cell tube formation. Collectively, these data indicate that chicken PBMSCs secrete bioactive factors with antimicrobial and regenerative properties, and as such, provide a novel source of cell-based therapies for the poultry industry.

Induced mammary cancer in rat models: pathogenesis, genetics, and relevance to female breast cancer.

Mammary cancer, or breast cancer in women, is a polygenic disease with a complex etiopathogenesis. While much remains elusive regarding its origin, it is well established that chemical carcinogens and endogenous estrogens contribute significantly to the initiation and progression of this disease. Rats have been useful models to study induced mammary cancer. They develop mammary tumors with comparable histopathology to humans and exhibit differences in resistance or susceptibility to mammary cancer depending on strain. While some rat strains (e.g., Sprague-Dawley) readily form mammary tumors following treatment with the chemical carcinogen, 7,12-dimethylbenz[a]-anthracene (DMBA), other strains (e.g., Copenhagen) are resistant to DMBA-induced mammary carcinogenesis. Genetic linkage in inbred strains has identified strain-specific quantitative trait loci (QTLs) affecting mammary tumors, via mechanisms that act together to promote or attenuate, and include 24 QTLs controlling the outcome of chemical induction, 10 QTLs controlling the outcome of estrogen induction, and 4 QTLs controlling the outcome of irradiation induction. Moreover, and based on shared factors affecting mammary cancer etiopathogenesis between rats and humans, including orthologous risk regions between both species, rats have served as useful models for identifying methods for breast cancer prediction and treatment. These studies in rats, combined with alternative animal models that more closely mimic advanced stages of breast cancer and/or human lifestyles, will further improve our understanding of this complex disease.

Comparative Efficacy of Topical Ophthalmic Ganciclovir and Oral Famciclovir in Cats with Experimental Ocular Feline Herpesvirus-1 Epithelial Infection.

Purpose: To determine the comparative efficacy of ganciclovir ophthalmic gel and famciclovir oral tablets in cats with experimentally induced ocular feline herpesvirus-1 (FHV-1) epithelial infection. Methods: A randomized, placebo-controlled trial was performed using 16 nonvaccinated, specific pathogen-free cats with experimental FHV-1 infection induced by topical ocular inoculation. Cats received topical ganciclovir 0.15% ophthalmic gel (1 drop 3 times daily, n = 6 cats), oral famciclovir (90 mg/kg twice daily, n = 6), or topical artificial tear gel (1 drop 3 times daily, n = 4) for 14 days. Cats were monitored after inoculation for 30 days. Ophthalmic examinations were performed every 2 days and ocular disease scores calculated. In vivo confocal microscopy was performed, and corneal leukocyte infiltrates quantified. Ocular samples for FHV-1 quantitative polymerase chain reaction (qPCR) and virus isolation assays were collected every 3 days. Hemograms and serum biochemistry panels were performed at intervals. Results: Clinical ocular disease scores and corneal leukocyte infiltrates were significantly lower in the ganciclovir and famciclovir groups compared with placebo, but no significant differences were detected between the antiviral treatment groups. Ocular viral loads determined by qPCR were significantly lower in the ganciclovir group compared with the placebo group, but there were no significant differences between the other study groups. Hemograms and biochemistry panels were unremarkable. Conclusion: Topical application of ganciclovir gel 3 times daily was well-tolerated and displayed similar efficacy at reducing clinical ocular disease scores and corneal inflammation as twice daily oral famciclovir treatment in cats with experimental ocular FHV-1 infection.

Establishment and characterization of equine mammary organoids using a method translatable to other non-traditional model species.

Mammary organoid (MaO) models are only available for a few traditional model organisms, limiting our ability to investigate mammary gland development and cancer across mammals. This study established equine mammary organoids (EqMaOs) from cryopreserved mammary tissue, in which mammary tissue fragments were isolated and embedded into a 3D matrix to produce EqMaOs. We evaluated viability, proliferation and budding capacity of EqMaOs at different time points during culture, showing that although the number of proliferative cells decreased over time, viability was maintained and budding increased. We further characterized EqMaOs based on expression of stem cell, myoepithelial and luminal markers, and found that EqMaOs expressed these markers throughout culture and that a bilayered structure as seen in vivo was recapitulated. We used the milk-stimulating hormone prolactin to induce milk production, which was verified by the upregulation of milk proteins, most notably ß-casein. Additionally, we showed that our method is also applicable to additional non-traditional mammalian species, particularly domesticated animals such as cats, pigs and rabbits. Collectively, MaO models across species will be a useful tool for comparative developmental and cancer studies.

The equine mesenchymal stromal cell secretome inhibits equid herpesvirus type 1 strain Ab4 in epithelial cells.

Equid herpesvirus 1 (EHV-1) outbreaks occur when virus spreads from infected horses to in-contact horses, primarily via nasal shedding. This study evaluated the efficacy of factors secreted by equine peripheral blood derived mesenchymal stromal cells (PB-MSCs), collectively named the secretome, to inhibit the growth of EHV-1 in (i) 2D epithelial cell cultures (RK-13) in vitro, (ii) 3D equine nasal explants in vitro and (iii) an EHV-1 infection mouse model in vivo. The PB-MSC secretome was found to inhibit EHV-1 in RK-13 cells as well as in the epithelium of equine nasal explants. Although the PB-MSC secretome did not decrease overall severity of EHV-1 infection in mice, as determined by weight loss and viral titers in lungs, histological analyses indicated local reduction of EHV-1 infection in nasal epithelium. These results indicate that the PB-MSC secretome inhibits EHV-1 in epithelial cells in a context-dependent manner.

Mesenchymal stromal cell-secreted CCL2 promotes antibacterial defense mechanisms through increased antimicrobial peptide expression in keratinocytes.

Mesenchymal stromal cells (MSCs) from both humans and horses, which represent a clinically relevant translation animal model for human cutaneous wound healing, were recently found to possess antimicrobial properties against planktonic bacteria, and in the case of equine MSCs, also against biofilms. This, together with previous findings that human and equine MSCs promote angiogenesis and wound healing, makes these cells an attractive approach to treat infected cutaneous wounds in both species. The anti-biofilm activities of equine MSC, via secretion of cysteine proteases, have only been demonstrated in vitro, thus lacking information about in vivo relevance. Moreover, the effects of the equine MSC secretome on resident skin cells have not yet been explored. The goals of this study were to (a) test the efficacy of the MSC secretome in a physiologically relevant ex vivo equine skin biofilm explant model and (b) explore the impact of the MSC secretome on the antimicrobial defense mechanisms of resident skin cells. Our salient findings were that secreted factors from equine MSCs significantly decreased viability of methicillin-resistant Staphylococcus aureus bacteria in mature biofilms in this novel skin biofilm explant model. Moreover, we demonstrated that equine MSCs secrete CCL2 that increases the antimicrobial activity of equine keratinocytes by stimulating expression of antimicrobial peptides. Collectively, these data contribute to our understanding of the MSC secretome's antimicrobial properties, both directly by killing bacteria and indirectly by stimulating immune responses of surrounding resident skin cells, thus further supporting the value of MSC secretome-based treatments for infected wounds.

The Horse as a Model for the Study of Cutaneous Wound Healing.

Significance: Cutaneous wounds are a major problem in both human and equine medicine. The economic cost of treating skin wounds and related complications in humans and horses is high, and in both species, particular types of chronic wounds do not respond well to current therapies, leading to suffering and morbidity. Recent Advances: Conventional methods for the treatment of cutaneous wounds are generic and have not changed significantly in decades. However, as more is learned about the mechanisms involved in normal skin wound healing, and how failure of these processes leads to chronic nonhealing wounds, novel therapies targeting the specific pathologies of hard-to-heal wounds are being developed and evaluated. Critical Issues: Physiologically relevant animal models are needed to (1) study the mechanisms involved in normal and impaired skin wound healing and (2) test newly developed therapies. Future Directions: Similarities in normal wound healing in humans and horses, and the natural development of distinct types of hard-to-heal chronic wounds in both species, make the horse a physiologically relevant model for the study of mechanisms involved in wound repair. Horses are also well-suited models to test novel therapies. In addition, studies in horses have the potential to benefit veterinary, as well as human medicine.

Translational Animal Models Provide Insight Into Mesenchymal Stromal Cell (MSC) Secretome Therapy.

The therapeutic potential of the mesenchymal stromal cell (MSC) secretome, consisting of all molecules secreted by MSCs, is intensively studied. MSCs can be readily isolated, expanded, and manipulated in culture, and few people argue with the ethics of their collection. Despite promising pre-clinical studies, most MSC secretome-based therapies have not been implemented in human medicine, in part because the complexity of bioactive factors secreted by MSCs is not completely understood. In addition, the MSC secretome is variable, influenced by individual donor, tissue source of origin, culture conditions, and passage. An increased understanding of the factors that make up the secretome and the ability to manipulate MSCs to consistently secrete factors of biologic importance will improve MSC therapy. To aid in this goal, we can draw from the wealth of information available on secreted factors from MSC isolated from veterinary species. These translational animal models will inspire efforts to move human MSC secretome therapy from bench to bedside.

Beyond tradition and convention: benefits of non-traditional model organisms in cancer research.

Traditional laboratory model organisms are indispensable for cancer research and have provided insight into numerous mechanisms that contribute to cancer development and progression in humans. However, these models do have some limitations, most notably related to successful drug translation, because traditional model organisms are often short-lived, small-bodied, genetically homogeneous, often immunocompromised, are not exposed to natural environments shared with humans, and usually do not develop cancer spontaneously. We propose that assimilating information from a variety of long-lived, large, genetically diverse, and immunocompetent species that live in natural environments and do develop cancer spontaneously (or do not develop cancer at all) will lead to a more comprehensive understanding of human cancers. These non-traditional model organisms can also serve as sentinels for environmental risk factors that contribute to human cancers. Ultimately, expanding the range of animal models that can be used to study cancer will lead to improved insights into cancer development, progression and metastasis, tumor microenvironment, as well as improved therapies and diagnostics, and will consequently reduce the negative impacts of the wide variety of cancers afflicting humans overall.

Single-cell RNA sequencing of equine mesenchymal stromal cells from primary donor-matched tissue sources reveals functional heterogeneity in immune modulation and cell motility.

BACKGROUND: The efficacy of mesenchymal stromal cell (MSC) therapy is thought to depend on the intrinsic heterogeneity of MSC cultures isolated from different tissue sources as well as individual MSCs isolated from the same tissue source, neither of which is well understood. To study this, we used MSC cultures isolated from horses. The horse is recognized as a physiologically relevant large animal model appropriate for translational MSC studies. Moreover, due to its large size the horse allows for the simultaneous collection of adequate samples from multiple tissues of the same animal, and thus, for the unique collection of donor matched MSC cultures from different sources. The latter is much more challenging in mice and humans due to body size and ethical constraints, respectively. METHODS: In the present study, we performed single-cell RNA sequencing (scRNA-seq) on primary equine MSCs that were collected from three donor-matched tissue sources; adipose tissue (AT), bone marrow (BM), and peripheral blood (PB). Based on transcriptional differences detected with scRNA-seq, we performed functional experiments to examine motility and immune regulatory function in distinct MSC populations. RESULTS: We observed both inter- and intra-source heterogeneity across the three sources of equine MSCs. Functional experiments demonstrated that transcriptional differences correspond with phenotypic variance in cellular motility and immune regulatory function. Specifically, we found that (i) differential expression of junctional adhesion molecule 2 (JAM2) between MSC cultures from the three donor-matched tissue sources translated into altered cell motility of BM-derived MSCs when RNA interference was used to knock down this gene, and (ii) differences in C-X-C motif chemokine ligand 6 (CXCL6) expression in clonal MSC lines derived from the same tissue source correlated with the chemoattractive capacity of PB-derived MSCs. CONCLUSIONS: Ultimately, these findings will enhance our understanding of MSC heterogeneity and will lead to improvements in the therapeutic potential of MSCs, accelerating the transition from bench to bedside.

Secreted sphingomyelins modulate low mammary cancer incidence observed in certain mammals.

Determining mechanisms that naturally protect species from developing cancer is critical in order to prevent and treat cancer. Here, we describe a novel cancer-suppressing mechanism, via the secretion of bioactive factors by mammary cells, that is present in domesticated mammals with a low mammary cancer incidence. Specifically, these bioactive factors induced triple-negative breast cancer cell (TNBC) death in vitro and reduced tumorigenicity in a xenograft TNBC mouse model in vivo. RNA deep sequencing showed significant downregulation of genes associated with breast cancer progression in secretome-cultured TNBC cells. Further in-depth multi-omics analysis identified sphingomyelins as key secreted factors, and their role was confirmed via inhibition of the sphingomyelin signaling pathway. We speculate that secreted sphingomyelins in the mammary gland of mammals with a naturally low incidence of mammary cancer mediate the elimination of cancer cells. This study contributes to the growing list of protective mechanisms identified in cancer-proof species.