Introduction Primary breast cancer surgery can compromise aesthetics and quality-of-life for breast cancer patients. While breast reconstruction improves these outcomes, current methods are limited by suboptimal aesthetic outcomes and potential complication risks. There is an urgent clinical need for improved approaches to post surgical reconstruction for breast cancer patients. Adipose-derived stromal cells (ADSCs) with biological scaffolds are being widely evaluated for tissue engineering applications in the field of reconstruction. Aims This study aimed to assess the biomechanical properties, biocompatibility, adipogenic potential of ADSCs encapsulated in modified hyaluronic acid derivatives in vitro; and efficacy and tissue integration of this construct in vivo in a murine breast cancer and reconstruction model. Methods ADSCs were obtained, with informed consent, from female breast cancer patients undergoing autologous breast reconstruction or cosmetic procedures (n=8) aged 47±12 years. Modified hyaluronic acid solution was combined with 1×106 ADSCs/mL and crosslinked using hydrogen peroxide and horseradish peroxidase. Young’s modulus, cell viability and adipogenic potential of the cell-loaded hydrogels were assessed in vitro. In vivo, hydrogels combined with murine ADSCs were grafted into a murine breast cancer model and tissues were harvested for immunohistochemistry after 4 weeks. Results ADSCs were characterised via morphology, Colony forming unit-fibroblast (CFU-F) assay, flow cytometry and multilineage differentiation. The cell-loaded hydrogels had a compressive Young’s modulus of 7.35±0.96 kPa after 21 days in culture, similar to human breast adipose tissue (∼10 kPa). High ADSC viability was observed after 21 days in culture, and ADSCs differentiated into mature adipocytes. After 4 weeks in vivo, hydrogels exhibited adipocytes, vascular endothelium, and pericyte-like cells. Conclusion This study demonstrates the potential suitability of modified hyaluronic acid hydrogels encapsulating ADSCs for adipose tissue engineering for post breast cancer reconstruction. ### Competing Interest Statement The authors have declared no competing interest.

Cancer cachexia is a highly prevalent wasting syndrome in cancer patients. Inflammation is hallmarks of symptomatic cachexia, however early stages of cachexia are not well understood, including differences between biological sexes. In a mouse model of early cachexia, muscle from males showed strong mitochondrial defects, whereas females were characterized by inflammatory and stress response. We demonstrate a novel link between the increase in purinergic receptor P26Y, and dysregulated Ca2+ homeostasis, mitochondrial dysfunction and damage, and inflammation during early stages of cancer cachexia. Low levels of miR-379-3p were associated with poor survival of patients with lung cancer. Restoring miR-379-3p levels in mice prevented loss of muscle mass and function. miR-379-3p targeted P2r6y and restored mitochondrial content and function, inhibited type II interferon response, and regulated the expression of Ca2+-related and apoptotic markers. This supports miR-379-3p as a hub regulating multiple processes underlying cachexia and represent a therapeutic target for cancer patients. ### Competing Interest Statement The authors have declared no competing interest.

Extracellular vesicles (EVs) are versatile transporters of genetic cargo with enormous potential in the therapeutic setting. Scalable production of EVs, and routes to overcome rapid clearance are required. Biocompatible hydrogels may support precise, localized delivery of EVs to target sites. This study aimed to establish sustained production of EVs in a scalable 3D dynamic bioreactor and to fabricate hydrogels using tyramine-modified hyaluronic acid (HA-TA) to study EV integration and release patterns. MDA-MB-231 cells transduced with lentiviral GFP fused with CD63, were cultured in a 20kD dynamic hollow fiber bioreactor and GFP-EVs harvested over five weeks. GFP-EVs were characterized by Nanoparticle Tracking Analysis(NTA), Western Blot(WB) and Transmission Electron Microscopy(TEM). Tyramine modified hyaluronic acid(HA-TA) hydrogels were formulated via enzymatic crosslinking using hydrogen peroxide and horseradish peroxidase, to investigate EV release patterns in static and dynamic conditions. Hydrogel swelling was recorded at 1-72 hrs and hydrogels were loaded with GFP-EVs to assess distribution and release by Scanning Electron Microscopy(SEM) and NTA respectively. GFP-EV uptake was assessed by confocal microscopy. Longitudinal GFP expression was demonstrated in transduced cells and released EVs throughout bioreactor culture. TEM and NTA demonstrated successful isolation of EVs of 30-200 nm in size with intact lipid bilayers (average 4x109 EVs/harvest). Initial harvests exhibited subpopulations of larger EVs, which disappeared upon serum withdrawal. WB verified the presence of EV markers CD63, TSG101, and CD81. HA-TA hydrogels were successfully formed and swelling assays revealed the requirement for higher concentrations of HA-TA and crosslinkers for scaffold stability and continued swelling. GFP-EVs were successfully incorporated into the hydrogels with variable release patterns observed over time, depending on EV concentration and hydrogel formulation. EV clusters in hydrogels were visualized by SEM. Investigation of GFP-EV release patterns under static and dynamic conditions highlighted a significant increase in release under fluid flow conditions. Efficient transfer of released EVs to recipient cells was also demonstrated in vitro. The data demonstrate the potential for scalable production of engineered EVs in serum free conditions and subsequent incorporation into HA-TA hydrogels for sustained release. These biocompatible hydrogels hold promise for tuneable delivery of therapeutic EVs in a variety of disease settings. ### Competing Interest Statement The authors have declared no competing interest.

Mesenchymal Stromal Cell derived extracellular vesicles (MSC-EVs) may retain the cancer targeting and immune privilege of MSCs. The immense potential MSC-EVs hold as tumour-targeted therapeutics warrants an understanding of potential adverse events to support clinical translation. This study aimed to determine whether MSC-EVs would elicit an immune response following administration in tumour-bearing immunocompetent animals. Secreted EVs were isolated from both human and murine bone marrow derived MSCs and characterized. hMSC-EVs or mMSC-EVs were administered intravenously into 4T1 breast tumour-bearing Balb/c mice or healthy controls. Tumour tissue, draining lymph nodes and spleens were harvested, dissociated into a single cell suspension and flow cytometry performed targeting T cells, myeloid derived suppressor cells (MDSCs), macrophages, dendritic cells and natural killer (NK) cells. The 4T1 model immune profile was first determined by comparing the spleen of tumour-bearing animals to healthy controls. T cells were increased in tumour-bearing animals (CD4+/CD25+ p=0.041; CD8+/CD25+ p=0.02). A significant elevation of GR-1+ MDSCs (p=0.002), CD11b+ macrophages (p=0.023) and CD11c+ dendritic cells (p=0.001) was also observed. In contrast, CD27+ NK cells were significantly decreased compared to the spleen of healthy animals (p=0.006). Collectively this data validated the immune profile and supported the determination of any changes in response to hMSC-EVs or mMSC-EVs administration. No significant activation of CD4+ (p=0.20) or CD8+ (p=0.57) T cells were seen in tumour tissue in both groups. The percentage of GR-1+ MDSCs (28% vs 27%, p=0.92), CD11b+, CD11c+ and CD27+ cells were similar regardless of EV origin. No significant changes in T cells, MDSCs, macrophages, dendritic or NK cells were observed in the lymph node or spleen of animals that received hMSC-EV versus mMSC-EVs. In conclusion, human MSC-EVs elicited no discernible immune response in mice, supporting the hypothesis that MSC-EVs retain the immune privilege of the secretory cell. This reinforces the therapeutic potential of MSC-EVs. ### Competing Interest Statement The authors have declared no competing interest.

Metallacarboranes have long been the subject of attention in the context of medicinal chemistry because of their promising characteristics and unconventional interactions with biological entities. The metal centre has been shown to have a significant influence on the internalisation and cytotoxicity of compounds in human cell lines. Additionally, specific nanomolar concentrations of metallacarboranes have demonstrated toxic effects on MDA-MB-231 cells under in vitro and in vivo conditions. ### Competing Interest Statement The authors have declared no competing interest.

Cobalt-based metallacarboranes have emerged as potential candidates for cancer treatment owing to their unique structural properties. In this study, a biocompatible delivery platform is developed by noncovalently incorporating cobalt metallacarborane (CoSAN) into hyaluronic acid (HA) functionalized with lysine (Lys). HA-Lys 2 enables the electrostatic interaction of CoSAN while retaining its cytotoxic activity, as confirmed by cellular assays using MDA-MB-231 triple-negative breast cancer cells. Elemental mapping via energy-dispersive X-ray spectroscopy (EDX) confirms the successful and homogeneous incorporation of CoSAN to lead HA-Lys-CoSAN 3, and the composite is further characterized using diffusion-ordered nuclear magnetic resonance (NMR) spectroscopy (DOSY). Stimulated Raman scattering (SRS) microscopy data demonstrate comparable cellular uptake in MDA-MB-231 cells of free and HA-loaded CoSAN. Additionally, release studies under physiologically relevant conditions show a sustained release profile over 24 h with pH dependency to mimic normal and tumor microenvironments. The present study describes a viable method for integrating metallacarboranes into a polymeric drug delivery system without compromising their anticancer properties, thereby advancing their potential for future therapeutic use.

Bone cells contribute to tumour metastasis by producing biochemical factors that stimulate tumour cell homing and proliferation, but also by resorbing bone matrix (osteolysis) that releases further stimulatory factors for tumour growth in a vicious cycle. Changes in the local mechanical environment of bone tissue occur during early metastasis, which might activate mechanobiological responses by resident bone cells (osteocytes) to activate resorption (osteoclasts) and thereby contribute to tumour invasion. The objective of this study is to investigate whether bone osteolysis is driven by early changes in the bone mechanical environment during metastasis by (a) implementing subject-specific FE models of metastatic femora to predict the mechanical environment within bone tissue during early metastasis (3-weeks after tumour inoculation) and then (b) applying mechanoregulation theory to predict bone tissue remodelling as a function of the evolving mechanical environment within bone tissue during breast cancer-bone metastasis. We implemented a global resorption rate derived from an experimental model, but the mechanoregulation algorithm predicted localised bone loss in the greater trochanter region, the same region where osteolysis was prevalent after three weeks of metastasis development in the animal model. Moreover, the mechanical environment evolved in a similar manner to that reported in separate subject-specific finite element models of these same animals by 6 weeks. Thus, we propose that early changes in the physical environment of bone tissue during metastasis may elicit mechanobiological cues for bone cells and activate later osteolytic bone destruction.

Cervical cancer is the fourth most frequently occurring cancer among women worldwide with 90 % of the new cases and deaths occurring in low and middle income countries [ [1] Sung H. Ferlay J. Siegel R.L. Laversanne M. Soerjomataram I. Jemal A. et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA Cancer J Clin. 2021; 71: 209-249 Crossref PubMed Scopus (43863) Google Scholar ]. In the 1970 s, cervical cancer accounted for 4 % of all deaths from cancer in women in England and Wales each year [ [2] Anderson M.C. 7 - The Aetiology and Pathology of Cancer of the Cervix. Clin Obstet Gynaecol. 1976; 3: 317-337 Crossref PubMed Google Scholar ]. Despite its prevalence there was limited understanding of the cause[ [2] Anderson M.C. 7 - The Aetiology and Pathology of Cancer of the Cervix. Clin Obstet Gynaecol. 1976; 3: 317-337 Crossref PubMed Google Scholar ]. Sexual intercourse was established to be a major risk factor by Gagnon and Towne [ 3 Gagnon F. Contribution to the study of the etiology and prevention of cancer of the cervix of the uterus. Am J Obstet Gynecol. 1950; 60: 516-522 Abstract Full Text PDF PubMed Scopus (78) Google Scholar , 4 Towne J.E. Carcinoma of the cervix in nulliparous and celibate women. Am J Obstet Gynecol. 1955; 69: 606-613 Abstract Full Text PDF PubMed Scopus (28) Google Scholar ]. Their studies investigating the medical records of nuns demonstrated how rare cervical cancer was in women assumed to never have had sexual intercourse [ 3 Gagnon F. Contribution to the study of the etiology and prevention of cancer of the cervix of the uterus. Am J Obstet Gynecol. 1950; 60: 516-522 Abstract Full Text PDF PubMed Scopus (78) Google Scholar , 4 Towne J.E. Carcinoma of the cervix in nulliparous and celibate women. Am J Obstet Gynecol. 1955; 69: 606-613 Abstract Full Text PDF PubMed Scopus (28) Google Scholar ].

Abstract Breast cancer metastasises to bone in 70–80% of patients with advanced disease. Bone cells contribute to tumour metastasis by activating bone resorption, which releases biochemical factors that stimulate tumour cell proliferation. The local mechanical environment of bone tissue is altered during early metastasis, prior to the formation of overt osteolytic metastasis. According to mechanoregulation theory, these changes might activate mechanobiological responses in bone cells and thereby contribute to osteolytic resorption. However, whether mechanobiological responses of bone cells drive osteolysis during metastasis is unknown. The objective of this study was to apply a computational mechanoregulation framework to predict how early changes in the bone mechanical environment contribute to osteolysis. Subject-specific finite element (FE) models were developed to predict the mechanical environment within bone tissue during early stage metastasis (3 weeks post-inoculation). We then applied a mechanoregulation algorithm to predict changes in bone tissue density as a function of the evolving mechanical environment due to tumour invasion. Substantial bone loss was predicted in the greater trochanter region, which coincides with experimental reports of regional bone loss in this animal model. Moreover, application of the mechanoregulation algorithm predicted that the mechanical environment evolved in a similar manner to that predicted through subject-specific finite element (FE) models. This is the first study to implement a computational mechanoregulation framework to predict the development of osteolysis. Our findings support the hypothesis that early changes in the physical environment of bone tissue during metastasis may elicit mechanobiological cues for bone cells and activate osteolytic destruction.

Abstract Backgorund Prior data suggest pre‐diagnostic aspirin use impacts breast tumour biology and patient outcome. Here, we employed faithful surgical resection models of HER2+ and triple‐negative breast cancer (TNBC), to study outcome and response mechanisms across breast cancer subtypes. Method NOD/SCID mice were implanted with HER2+ MDA‐MB‐231/LN/2‐4/H2N, trastuzumab‐resistant HER2+ HCC1954 or a TNBC patient‐derived xenograft (PDX). A daily low‐dose aspirin regimen commenced until primary tumours reached ~250 mm3 and subsequently resected. MDA‐MB‐231/LN/2‐4/H2N mice were monitored for metastasis utilising imaging. To interrogate the survival benefit of pre‐treatment aspirin, 3 weeks post‐resection, HCC1954/TNBC animals received standard‐of‐care (SOC) chemotherapy for 6 weeks. Primary tumour response to aspirin was interrogated using immunohistochemistry. Results Aspirin delayed time to metastasis in MDA‐MB‐231/LN/2‐4/H2N xenografts and decreased growth of HER2+/TNBC primary tumours. Lymphangiogenic factors and lymph vessels number were decreased in HER2+ tumours. However, no survival benefit was seen in aspirin pre‐treated animals (HCC1954/TNBC) that further received adjuvant SOC, compared with animals treated with SOC alone. In an effort to study mechanisms responsible for the observed reduction in lymphangiogenesis in HER2+ BC we utilised an in vitro co‐culture system of HCC1954 tumour cells and mesenchymal stromal cells (MSC). Aspirin abrogated the secretion of VEGF‐C in MSCs and also decreased the lymph/angiogenic potential of the MSCs and HCC1954 by tubule formation assay. Furthermore, aspirin decreased the secretion of uPA in HCC1954 cells potentially diminishing its metastatic capability. Conclusion Our data employing clinically relevant models demonstrate that aspirin alters breast tumour biology. However, aspirin may not represent a robust chemo‐preventative agent in the HER2+ or TNBC setting.

Extracellular vesicles (EVs) are nanoparticles found in all biological fluids, capable of transporting biological material around the body. Extensive research into the physiological role of EVs has led to the development of the Minimal Information for Studies of Extracellular Vesicles (MISEV) framework in 2018. This framework guides the standardisation of protocols in the EV field. To date, the focus has been on EVs of human origin. As comparative medicine progresses, there has been a drive to study similarities between diseases in humans and animals. To successfully research EVs in felines, we must validate the application of the MISEV guidelines in this group. EVs were isolated from the plasma of healthy humans and felines. EV characterisation was carried out according to the MISEV guidelines. Human and feline plasma showed a similar concentration of EVs, comparable expression of known EV markers and analogous particle to protein ratios. Mass spectrometry analyses showed that the proteomic signature of EVs from humans and felines were similar. Asymmetrical flow field flow fractionation, showed two distinct subpopulations of EVs isolated from human plasma, whereas only one subpopulation was isolated from feline plasma. Metabolomic profiling showed similar profiles for humans and felines. In conclusion, isolation, and characterisation of EVs from humans and felines show that MISEV2018 guidelines may also be applied to felines. Potential comparative medicine studies of EVs may provide a model for studying naturally occurring diseases in both humans and felines.

BACKGROUND AND AIM:Adipose-derived stromal cells (ASCs) are a promising cell source for novel tissue engineering approaches to breast reconstruction following cancer resection. However there is limited knowledge on the effect of adjuvant therapies such as hormonal therapy on ASCs, which may affect their efficacy in regenerative strategies. The present study aims to investigate the effects of Tamoxifen and its metabolites Afimoxifene (4-Hydroxy-Tamoxifen) and Endoxifen (N-desmethyl-4-hydroxytamoxifen) on patient-derived ASC viability, apoptosis, adipogenic differentiation and angiogenic potential.METHODS:ASCs were isolated from fat harvested from female breast cancer patients undergoing breast reconstruction surgery or cosmetic procedures. Oestrogen receptor (ER α, β) expression was analysed using immunofluorescence. ASCs were then treated with various concentrations of Afimoxifene, Endoxifen and Tamoxifen (combination), and the impact on ASC viability and apoptosis determined. ASCs were cultured in adipogenic-differentiation media with or without tamoxifen and derivatives, and adipogenesis was measured using quantitative Real-time Polymerase chain reaction (qRT-PCR) and histological staining (Oil Red O). The effect on secreted VEGF levels was also quantified in ASC conditioned media RESULTS: ASCs were successfully isolated and characterised from human abdominal lipoaspirates or fat tissues (n = 8). ASCs subjected to varying doses of Tamoxifen and metabolites (up to 1000 nM) showed no decline in cell viability or increase in apoptosis, at physiological doses (upto 100 nM). Functional decline in adipogenic differentiation or gene expression was observed at supraphysiological concentrations of Tamoxifen (1000 nM). VEGF165 protein secretion in ASC-cell conditioned media was not significantly impacted irrespective of dosage.CONCLUSION:At physiologically relevant doses, Tamoxifen treatment did not result in any deleterious effect on ASC survival and functionality and is unlikely to negatively impact ASC based breast reconstruction strategies for breast cancer patients receiving this adjuvant hormonal therapy.