Transcriptomics of Canine Inflammatory Mammary Cancer Treated with Empty Cowpea Mosaic Virus Implicates Neutrophils in Anti-Tumor Immunity.

Canine inflammatory mammary cancer (IMC) is a highly aggressive and lethal cancer in dogs serving as a valuable animal model for its human counterpart, inflammatory breast cancer (IBC), both lacking effective therapies. Intratumoral immunotherapy (IT-IT) with empty cowpea mosaic virus (eCPMV) nanoparticles has shown promising results, demonstrating a reduction in tumor size, longer survival rates, and improved quality of life. This study compares the transcriptomic profiles of tumor samples from female dogs with IMC receiving eCPMV IT-IT and medical therapy (MT) versus MT alone. Transcriptomic analyses, gene expression profiles, signaling pathways, and cell type profiling of immune cell populations in samples from four eCPMV-treated dogs with IMC and four dogs with IMC treated with MT were evaluated using NanoString Technologies using a canine immune-oncology panel. Comparative analyses revealed 34 differentially expressed genes between treated and untreated samples. Five genes (CXCL8, S100A9, CCL20, IL6, and PTGS2) involved in neutrophil recruitment and activation were upregulated in the treated samples, linked to the IL17-signaling pathway. Cell type profiling showed a significant increase in neutrophil populations in the tumor microenvironment after eCPMV treatment. These findings highlight the role of neutrophils in the anti-tumor response mediated by eCPMV IT-IT and suggest eCPMV as a novel therapeutic approach for IBC/IMC.

Epitheliosis is a histopathological finding associated with malignancy and poor prognosis in dogs with mammary tumors.

Canine mammary epitheliosis (ME) is a poorly studied dysplasia that may have premalignant potential. In this study, the clinicopathological relevance of ME was prospectively studied in 90 female dogs with mammary tumors (MTs) that underwent radical mastectomy. ME distribution, extent, and coexistence with benign and malignant MTs were evaluated for each case (505 mammary glands). ME was macroscopically undetectable and was present in 47/90 (52%) cases, frequently bilateral. In dogs with malignant MTs and ME, diffuse ME throughout the mammary chain was present in 10/39 (26%) cases. A histological ME-carcinoma transition was evident in certain histotypes. By immunohistochemistry (AE1/AE3, cytokeratin 14 [CK-14], CK-8/18, vimentin, calponin, p63, Ki-67, estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2), ME was a slow-growing, triple-negative process with a strong predominance of basal-like nonmyoepithelial cells. ME was associated with older dogs (P = .016), malignant tumors (P = .044), worse clinical stages (P = .013), lymph node metastasis (LNM, P = .021), higher histological grade tumors (P = .035), and shorter overall survival (OS) in univariate analysis (P = .012). Interestingly, ME was distantly located to the malignant tumor in most cases (P = .007). In multivariate analyses, LNM (P = .005), histological grade (P = .006), and tumor size (P = .006) were independent predictors of OS. For the pathologist, the observation of ME should be clearly stated in the MT biopsy report to alert the surgeon/oncologist. Given the differences between canine ME and its human histopathological counterpart (atypical ductal hyperplasia), "epitheliosis" should remain the preferred term for the dog.

Effects of doxorubicin associated with amniotic membrane stem cells in the treatment of canine inflammatory breast carcinoma (IPC-366) cells.

BACKGROUND: Tumours in mammary glands represent the most common neoplasia in bitches, as in humans. This high incidence results in part from the stimulation of sex hormones on these glands. Among mammary tumours, inflammatory carcinoma is the most aggressive, presenting a poor prognosis to surgical treatment and chemotherapy. One of the most widely used chemotherapy drugs for breast cancer treatment is doxorubicin (DOXO). Alternative therapies have been introduced in order to assist in these treatments; studies on treatments using stem cells have emerged, since they have anti-inflammatory and immunomodulatory properties. The aim of this study was to evaluate the effects of DOXO and canine amniotic membrane stem cells (AMCs) on the triple-negative canine inflammatory mammary carcinoma cell line IPC-366. METHODS: Four experimental groups were analysed: a control group without treatment; Group I with DOXO, Group II with AMC and Group III with an association of DOXO and AMCs. We performed the MTT assay with DOXO in order to select the best concentration for the experiments. The growth curve was performed with all groups (I-III) in order to verify the potential of treatments to reduce the growth of IPC-366. For the cell cycle, all groups (I-III) were tested using propidium iodide. While in the flow cytometry, antibodies to progesterone receptor (PR), estrogen receptor (ER), PCNA, VEGF, IL-10 and TGF-ß1 were used. For steroidogenic pathway hormones, an ELISA assay was performed. RESULTS: The results showed that cells treated with 10 µg/mL DOXO showed a 71.64% reduction in cellular growth after 72 h of treatment. Reductions in the expression of VEGF and PCNA-3 were observed by flow cytometry in all treatments when compared to the control. The intracellular levels of ERs were also significantly increased in Group III (4.67% vs. 27.1%). Regarding to the levels of steroid hormones, significant increases in the levels of estradiol (E2) and estrone sulphate (S04E1) were observed in Groups I and III. On the other hand, Group II did not show differences in steroid hormone levels in relation to the control. We conclude that the association of DOXO with AMCs (Group III) promoted a reduction in cell growth and in the expression of proteins related to proliferation and angiogenesis in IPC-366 triple-negative cells. CONCLUSIONS: This treatment promoted ER positive expression, suggesting that the accumulated oestrogen conducted these cells to a synergistic state, rendering these tumour cells responsive to ERs and susceptible to new hormonal cancer therapies.

Vasculogenic mimicry-associated ultrastructural findings in human and canine inflammatory breast cancer cell lines.

BACKGROUND: Human inflammatory breast cancer (IBC) and canine inflammatory mammary cancer (IMC) are the most lethal mammary cancers. An exacerbated angiogenesis and the existence of vasculogenic mimicry (VM) are hallmarks of these tumors. The information regarding VM and ultrastructural characteristics of mammary cell lines is scant. METHODS: In this study, IBC cell line SUM149 and IMC cell line IPC-366 in adherent (2D) and non-adherent (3D) (mammospheres, cancer stem cells) conditions were analyzed by transmission and scanning electron microscopy (TEM and SEM, respectively). RESULTS: The TEM revealed round to oval shape cells with microvilli on the surface, high numbers of peroxisomes in close apposition to lipid droplets and some extracellular derived vesicles. The TEM and the SEM mammospheres revealed group of cells clumping together with a central lumen (resembling a mammary acini). The cells joint are tight junctions and zonula adherens. By SEM two cell morphologies were observed: spherical and flattened cells. There was evidence endothelial-like cells (ELCs), which is characteristic for this disease, showing several or unique cytoplasmic empty space. ELCs were more frequent in 3D than in 2D culture conditions and contained Weibel-Palade cytoplasmic bodies, which are exclusive structures of endothelial cells. CONCLUSIONS: Both cell lines, IPC-366 and SUM-149, shared ultrastructural characteristics, further supporting canine IMC as a model for the human disease. To the best of our knowledge, this is the first study that demonstrate the morphological differentiation of cultured cancer stem cells from cancer epithelial cell lines into endothelial-like cells, confirming the vasculogenic mimicry phenomenon from an ultrastructural point of view.

Canine Spindle Cell Mammary Tumor: A Retrospective Study of 67 Cases.

Canine spindle cell mammary tumor (CSCMT) is an infrequent canine mammary tumor (CMT) composed of spindle or fusiform cells, which represents a challenge for pathologists and clinicians. Mammary tumors submitted for histopathology from 1998 to 2013 and compatible with CSCMTs were retrospectively selected. The tumors were diagnosed based on the hematoxylin and eosin (HE)-stained section; malignant tumors were graded using a canine soft tissue sarcoma grading scheme and a canine mammary tumor grading scheme, and they were further assigned a diagnosis based on immunohistochemistry (IHC) for pancytokeratin, cytokeratin 14, p63, calponin, vimentin, Ki-67, CD31, desmin, myosin, smooth muscle actin, glial fibrillary acidic protein, and S-100. The origin of the tumors was assessed as mammary, skin, or unknown. The prevalence of CSCMT was 1% of all CMTs. CSCMTs included 3 benign tumors (1 angioma and 2 benign myoepitheliomas) and 67 malignant tumors that after IHC were diagnosed as malignant myoepithelioma (64%), carcinoma and malignant myoepithelioma (19%), hemangiosarcoma (8%), undifferentiated sarcoma (5%), peripheral nerve sheath tumor (3%), and fibrosarcoma (2%). The diagnosis based on the HE-stained section differed from the diagnosis after IHC in 75% of the malignant cases. The majority of malignant CSCMTs were solitary (57%) large tumors (6.42 ± 3.92 cm) with low metastatic potential and high survival rate (8% tumor-related mortality). Higher sarcoma grade was associated with older age (P = .034) and greater tumor size (P = .037). Malignant CSCMTs need to be evaluated by IHC to ensure the histotype and the relatively benign clinical behavior, despite their large size.

P-Glycoprotein and Breast Cancer Resistance Protein in Canine Inflammatory and Noninflammatory Grade III Mammary Carcinomas.

P-glycoprotein (P-gp/ABCB1) and breast cancer resistance protein (BCRP/ABCG2) expression are frequently related to multidrug resistance (MDR) in neoplastic cells. Canine inflammatory and grade III noninflammatory mammary carcinomas (IMC and non-IMC) are aggressive tumors that could benefit from chemotherapy. This study describes the immunohistochemical detection of P-gp and BCRP in 20 IMCs and 18 non-IMCs from dogs that had not received chemotherapy. Our aim was to determine if P-gp and BCRP expression was related to the "inflammatory" phenotype, to establish a basis for future studies analyzing the response to chemotherapy in dogs with highly malignant mammary cancer. Immunolabeling was primarily membranous for P-gp with a more intense labeling in emboli, and immunolabeling was membranous and cytoplasmic for BCRP. P-gp was expressed in 17 of 20 (85%) IMCs compared to 7 of 18 (39%) non-IMCs (P = 0.006). BCRP was expressed within emboli in 15 of 19 (79%) emboli in IMC, 12 of 15 (80%) primary IMCs, and 12 of 18 (67%) non-IMCs, without statistically significant differences (P > .05). All IMCs and 67% of non-IMCs expressed at least 1 of the 2 transporters, and 63% (12/19) of IMCs and 39% (7/18) of non-IMCs expressed both P-gp and BCRP. P-gp and BCRP evaluation might help select patients for chemotherapy. P-gp, expressed in a significantly higher percentage of IMCs vs non-IMCs, might play a specific role in the chemoresistance of IMC.

Long-Term Exposure to Isoflavones Alters the Hormonal Steroid Homeostasis-Impairing Reproductive Function in Adult Male Wistar Rats.

The consumption of isoflavones is gaining popularity worldwide due to their beneficial effects on health. However, isoflavones are considered to be endocrine disruptors and cause deleterious effects on hormone-sensitive organs, especially in males. Therefore, this study aimed to determine if a continuous and prolonged exposure to isoflavones in adult males altered the endocrine axis effect of testicular function. For this purpose, seventy-five adult male rats were administered with low and high mixtures of isoflavones (genistein and daidzein) for 5 months. The determination of steroid hormones (progesterone, androstenedione, dehydroepiandrosterone, testosterone, dihydrotestosterone, 17ß-estradiol, and estrone sulphate) was carried out in serum and testicular homogenate samples. Sperm quality parameters and testicular histology were also determined. The results revealed that low and high doses of isoflavones promote a hormonal imbalance in androgen and estrogen production, resulting in a decrease in circulating and testicular androgen levels and an increase in estrogen levels. These results are associated with a reduction in the sperm quality parameters and a reduction in the testicular weight, both in the diameter of the seminiferous tubules and the height of the germinal epithelium. Altogether, these results suggest that a continuous exposure to isoflavones in adult male rats causes a hormonal imbalance in the testes that disrupts the endocrine axis, causing defects in testicular function.

Neoadjuvant Intratumoral Immunotherapy with Cowpea Mosaic Virus Induces Local and Systemic Antitumor Efficacy in Canine Mammary Cancer Patients.

The lack of optimal models to evaluate novel agents is delaying the development of effective immunotherapies against human breast cancer (BC). In this prospective open label study, we applied neoadjuvant intratumoral immunotherapy with empty cowpea mosaic virus-like particles (eCPMV) to 11 companion dogs diagnosed with canine mammary cancer (CMC), a spontaneous tumor resembling human BC. We found that two neoadjuvant intratumoral eCPMV injections resulted in tumor reduction in injected tumors in all patients and in noninjected tumors located in the ipsilateral and contralateral mammary chains of injected dogs. Tumor reduction was independent of clinical stage, tumor size, histopathologic grade, and tumor molecular subtype. RNA-seq-based analysis of injected tumors indicated a decrease in DNA replication activity and an increase in activated dendritic cell infiltration in the tumor microenvironment. Immunohistochemistry analysis demonstrated significant intratumoral increases in neutrophils, T and B lymphocytes, and plasma cells. eCPMV intratumoral immunotherapy demonstrated antitumor efficacy without any adverse effects. This novel immunotherapy has the potential for improving outcomes for human BC patients.

Clinical outcome of dogs diagnosed with canine inflammatory mammary cancer treated with metronomic cyclophosphamide, a cyclooxygenase-2 inhibitor and toceranib phosphate.

Canine inflammatory mammary cancer (IMC) is highly malignant, invasive and a therapeutic challenge, because effective medical treatment is still unavailable. This retrospective study compares the efficacy of an oral cyclooxygenase-2 (COX-2) inhibitor combined with toceranib phosphate and oral cyclophosphamide (multi-drug therapy [MT]) with COX-2 inhibitor therapy alone (single-drug therapy [ST]) in dogs diagnosed with secondary IMC. Clinical response, adverse events, overall survival time (OST), disease-free survival (DFS) and time to progression (TTP) were evaluated. Sixteen patients were included, eight received MT and eight receiving ST. Median OST was significantly higher in patients receiving MT (96.0 vs. 37.5 days; p = .046) and in patients with post-surgical rather than non-surgical IMC (86.5 vs. 41.5 days; p = .038). Additionally, median TTP was significantly higher in patients treated with MT (p = .010). In patients with non-surgical IMC, the clinical benefit (CB) was reached in 100% (n = 3) of patients receiving MT and in 33% (n = 1) of those receiving ST; the response duration was significantly longer in MT cases (p = .026). The absence of disease progression at day 30 of treatment was significantly associated with longer OST, DFS and TTP (p = .018, p = .002 and p < .001, respectively). Adverse events occurred more frequently in patients treated with MT compared with ST (p = .026). The MT protocol produced primarily mild to moderate toxicities, which were resolved with supportive care; therefore, the combination of drugs was adequately tolerated by most of the patients. The combination of toceranib, a COX-2 inhibitor and oral cyclophosphamide may be a protocol with potential therapeutic efficacy for dogs with IMC.

Neoadjuvant in situ vaccination with cowpea mosaic virus as a novel therapy against canine inflammatory mammary cancer.

BACKGROUND: Inflammatory mammary cancer (IMC), the counterpart of human inflammatory breast cancer (IBC), is the deadliest form of canine mammary tumors. IMC patients lack specific therapy and have poor outcomes. This proof-of-principle preclinical study evaluated the efficacy, safety, and effect on survival of neoadjuvant intratumoral (in situ) empty cowpea mosaic virus (eCPMV) immunotherapy in companion dogs diagnosed with IMC. METHODS: Ten IMC-bearing dogs were enrolled in the study. Five dogs received medical therapy, and five received weekly neoadjuvant in situ eCPMV immunotherapy (0.2-0.4 mg per injection) and medical therapy after the second eCPMV injection. Efficacy was evaluated by reduction of tumor growth; safety by hematological and biochemistry changes in blood and plasma; and patient outcome by survival analysis. eCPMV-induced immune changes in blood cells were analyzed by flow cytometry; changes in the tumor microenvironment were evaluated by CD3 (T lymphocytes), CD20 (B lymphocytes), FoxP3 (Treg lymphocytes), myeloperoxidase (MPO; neutrophils), Ki-67 (proliferation index, PI; tumor cell proliferation), and Cleaved Caspase-3 (CC-3; apoptosis) immunohistochemistry. RESULTS: Two neoadjuvant in situ eCPMV injections resulted in tumor shrinkage in all patients by day 14 without systemic adverse events. Although surgery for IMC is generally not an option, reduction in tumor size allowed surgery in two IMC patients. In peripheral blood, in situ eCPMV immunotherapy was associated with a significant decrease of Treg+/CD8+ ratio and changes in CD8+Granzyme B+ T cells, which behave as a lagging predictive biomarker. In the TME, higher neutrophilic infiltration and MPO expression, lower tumor Ki-67 PI, increase in CD3+ lymphocytes, decrease in FoxP3+/CD3+ ratio (p<0.04 for all comparisons), and no changes in CC-3+ immunostainings were observed in post-treatment tumor tissues when compared with pretreatment tumor samples. eCPMV-treated IMC patients had a statistically significant (p=0.033) improved overall survival than patients treated with medical therapy. CONCLUSIONS: Neoadjuvant in situ eCPMV immunotherapy demonstrated anti-tumor efficacy and improved survival in IMC patients without systemic adverse effects. eCPMV-induced changes in immune cells point to neutrophils as a driver of immune response. Neoadjuvant in situ eCPMV immunotherapy could be a groundbreaking immunotherapy for canine IMC and a potential future immunotherapy for human IBC patients.

From Conventional to Precision Therapy in Canine Mammary Cancer: A Comprehensive Review.

Canine mammary tumors (CMTs) are the most common neoplasm in intact female dogs. Canine mammary cancer (CMC) represents 50% of CMTs, and besides surgery, which is the elective treatment, additional targeted and non-targeted therapies could offer benefits in terms of survival to these patients. Also, CMC is considered a good spontaneous intermediate animal model for the research of human breast cancer (HBC), and therefore, the study of new treatments for CMC is a promising field in comparative oncology. Dogs with CMC have a comparable disease, an intact immune system, and a much shorter life span, which allows the achievement of results in a relatively short time. Besides conventional chemotherapy, innovative therapies have a large niche of opportunities. In this article, a comprehensive review of the current research in adjuvant therapies for CMC is conducted to gather available information and evaluate the perspectives. Firstly, updates are provided on the clinical-pathological approach and the use of conventional therapies, to delve later into precision therapies against therapeutic targets such as hormone receptors, tyrosine kinase receptors, p53 tumor suppressor gene, cyclooxygenases, the signaling pathways involved in epithelial-mesenchymal transition, and immunotherapy in different approaches. A comparison of the different investigations on targeted therapies in HBC is also carried out. In the last years, the increasing number of basic research studies of new promising therapeutic agents on CMC cell lines and CMC mouse xenografts is outstanding. As the main conclusion of this review, the lack of effort to bring the in vitro studies into the field of applied clinical research emerges. There is a great need for well-planned large prospective randomized clinical trials in dogs with CMC to obtain valid results for both species, humans and dogs, on the use of new therapies. Following the One Health concept, human and veterinary oncology will have to join forces to take advantage of both the economic and technological resources that are invested in HBC research, together with the innumerable advantages of dogs with CMC as a spontaneous animal model.

Anti-Angiogenic Treatments Interact with Steroid Secretion in Inflammatory Breast Cancer Triple Negative Cell Lines.

Human inflammatory breast cancer (IBC) is a highly angiogenic disease for which antiangiogenic therapy has demonstrated only a modest response, and the reason for this remains unknown. Thus, the purpose of this study was to determine the influence of different antiangiogenic therapies on in vitro and in vivo steroid hormone and angiogenic growth factor production using canine and human inflammatory breast carcinoma cell lines as well as the possible involvement of sex steroid hormones in angiogenesis. IPC-366 and SUM149 cell lines and xenotransplanted mice were treated with different concentrations of VEGF, SU5416, bevacizumab and celecoxib. Steroid hormone (progesterone, dehydroepiandrostenedione, androstenedione, testosterone, dihydrotestosterone, estrone sulphate and 17ß-oestradiol), angiogenic growth factors (VEGF-A, VEGF-C and VEGF-D) and IL-8 determinations in culture media, tumour homogenate and serum samples were assayed by EIA. In vitro, progesterone- and 17ß-oestradiol-induced VEGF production promoting cell proliferation and androgens are involved in the formation of vascular-like structures. In vivo, intratumoural testosterone concentrations were augmented and possibly associated with decreased metastatic rates, whereas elevated E1SO4 concentrations could promote tumour progression after antiangiogenic therapies. In conclusion, sex steroid hormones could regulate the production of angiogenic factors. The intratumoural measurement of sex steroids and growth factors may be useful to develop preventive and individualized therapeutic strategies.

Blocking Estrogen Synthesis Leads to Different Hormonal Responses in Canine and Human Triple Negative Inflammatory Breast Cancer.

Blocking estrogen synthesis by inhibitors of estrogen synthesis is a widely used therapy against estrogen receptor-positive tumors. However, these therapies are less effective in negative expression tumors. Therefore, this study determined the effectiveness of anti-aromatase and anti-sulfatase therapies in canine and human inflammatory breast cancer. Cell cultures and xenografts from IPC-366 and SUM149 were treated with different doses of letrozole (anti-aromatase) and STX-64 (anti-sulfatase), in order to observe their effectiveness in terms of cell proliferation, tumor progression, and the appearance of metastases and hormonal profiles. The results revealed that both treatments are effective in vitro since they reduce cell proliferation and decrease the secreted estrogen levels. In xenograft mice, while treatment with letrozole reduces tumor progression by 30-40%, STX-64 increases tumor progression by 20%. The hormonal results obtained determined that STX-64 produced an increase in circulating and intratumoral levels of estradiol, which led to an increase in tumor progression. However, letrozole was able to block estrogen synthesis by decreasing the levels of circulating and intratumoral estrogen and thus slowing down tumor progression. In conclusion, letrozole can be an effective treatment for canine and human inflammatory breast cancer. The knowledge of the hormonal profile of breast tumors reflects useful information on the effectiveness of different endocrine treatments.

Tumor Growth Progression in Ectopic and Orthotopic Xenografts from Inflammatory Breast Cancer Cell Lines.

Xenografts can grow in immunosuppressed hosts, such as SCID mice, and tumor material can be injected into hosts either ectopically or orthotopically. Choosing the correct model to use is a crucial step in animal research. The aim of this study was to report the differences between ectopic and orthotopic xenografts in tumor progression, metastasis capacity, histological features, and steroid hormone profiles in xenografts from the cIMC (canine inflammatory mammary cancer) cell line IPC-366 and hIBC (human inflammatory breast cancer) cell line SUM149. To achieve this purpose, 40 female mice 6-8 weeks old were inoculated with IPC-366 and SUM149 cells subcutaneously (ectopic models) or into mammary fat pad (orthotopic models). Mice were monitored for tumor progression and appearance of metastases, and generated tumors were analyzed in terms of histological examination and steroid hormone production. The results revealed differences in tumor appearance and percentage of metastasis between ectopic and orthotopic models, which were higher in the ectopic xenografts from both cell lines. However, both models had similar characteristics of tumor progression, histological features, and steroid hormone secretion profiles. We show that the ectopic model can be validated as a good and useful model of tumor development in addition to, not contrary to, the orthotopic model in breast cancer research.