Biology subjects

Branco, C. M.

Publications and source records attributed to Branco, C. M..

6 recordsLinked to original sources

bioRxiv2026

ORTHOTOPIC TUMOUR INDUCTION PROTOCOL SHAPES TUMOUR MICROENVIRONMENT

Robust preclinical cancer models are essential for studying tumour progression and evaluating therapeutic strategies. Orthotopic implantation of tumour cells into the mammary gland is widely used to model triple-negative breast cancer (TNBC), yet substantial variation exists in the number of cells used to initiate tumours, often without explicit consideration of how this parameter shapes tumour biology. Here, we systematically assess how tumour cell number at implantation influences the tumour microenvironment using the 4T1 orthotopic TNBC model. Mammary tumours initiated with different cell numbers exhibited comparable early growth kinetics and gross size, yet diverged markedly in cellular density, hypoxia, necrosis, proliferation, vascular integrity, and immune cell infiltration. High-density tumours displayed increased HIF-1 staining and necrosis, alongside differences in vascular-associated markers and increased macrophage recruitment. Conversely, low-density tumours remained highly proliferative, less necrotic and exhibited higher PDGFR{beta} staining. Importantly, tumour cells disseminated systemically within days of implantation across all conditions, although differences in colony recovery and ex vivo expansion were not statistically significant. Together, these findings demonstrate that tumour initiating protocols are powerful determinants of tumour state, with potential implications for dissemination frequency and tumour cell fate at distant sites. Rather than representing a source of experimental variability alone, this understanding provides an opportunity to intentionally tailor orthotopic models to investigate specific stages of disease progression and therapeutic response, thereby enhancing cross-study reproducibility and translational relevance.

cancer biology↗

bioRxiv2025

Pericyte-tumor crosstalk facilitates metastatic tumor cell latency through PIEZO1-activated lysophospholipid transfer.

Tumor dissemination is increasingly recognized to begin early in tumor development. Although most of these early disseminated cells are cleared, some survive and persist below clinical detection, acting as reservoirs for metastatic relapse. Metastatic tumor cells often rely on interactions with local stromal cells to support their colonization. In this study, we propose that pericyte-tumor cell interactions promote dormancy induction in the early metastatic lung, enhancing disseminated tumor cell (DTC) persistence. Extravital imaging demonstrated that DTCs interact with pericytes upon extravasation into the lung. Co-culture experiments were used to assess DTC fate after pericyte contact and revealed that transient contact with pericytes reduced the proliferation of metastatic 4T1 breast cancer cells but had no effect on non-metastatic 67NR cells. In vivo, transient pericyte contact resulted in higher lung metastatic burden, driven by small, non-proliferative lesions (<6 cells), 10 days after intracardiac injection. These lesions exhibited reduced KI67 staining and EdU incorporation compared to those from monocultured cells. We further observed that primary lung pericytes transferred lyso-phospholipids (lyso-PLs) specifically to metastatic 4T1 cells through direct contact. Gene expression analysis indicated that transient pericyte contact activated pathways related to syncytium formation in metastatic cells. In normal physiology, pericytes act in a syncytium to regulate blood flow via mechanosensitive channels in response to blood pressure changes. We hypothesize that tumor cells exploit these mechanosensitive responses to trigger lyso-PL transfer from pericytes. Supporting this, calcium imaging showed higher calcium activity in pericytes co-cultured with 4T1 cells, and calcium channel inhibitors significantly reduced lyso-PL transfer. Pharmacological activation of pericyte calcium channels induced lyso-PL release, which was subsequently taken up by tumor cells. Conditioned medium from activated pericytes, containing free lyso-PLs, recapitulated the reduced proliferation observed in transient co-culture. Finally, we found our pericyte-induced dormancy signature to be associated with tumor dormancy and distant metastasis free survival latency in breast cancer patients. Together, these findings suggest that early DTCs may exploit pericyte signaling mechanisms to enter dormancy, facilitating their persistence at metastatic sites and contributing to future relapse.

cancer biology↗

bioRxiv2024

Pericytes require physiological oxygen tension to maintain phenotypic fidelity.

Pericytes function to maintain tissue homeostasis by regulating capillary blood flow and maintaining endothelial barrier function. Pericyte dysfunction is associated with various pathologies and has recently been found to aid cancer progression. Despite having critical functions in health and disease, pericytes remain an understudied population due to a lack of model systems which accurately reflect in vivo biology. In this study we developed a protocol to isolate and culture murine lung, brain, bone, and liver pericytes, that maintains their known phenotypes and functions. We demonstrate that pericytes, being inherently plastic, benefit from controlled oxygen tension culture conditions, aiding their expansion ex vivo. Primary pericytes grown in physioxia (10% O2 for lung; 5% O2 for brain, bone, and liver) also better retain pericyte phenotypes indicated by stable expression of characteristic transcriptional and protein markers. In functional tube formation assays, pericytes were observed to significantly associate with endothelial junctions. Importantly, we identified growth conditions that limit expression of the plasticity factor Klf4 to prevent spontaneous phenotypic switching in vitro. Additionally, we were able to induce pathological pericyte phenotypic switching in response to metastatic stimuli to accurately recapitulate in vivo biology. Here, we present a robust method for studying pericyte biology in both physiology and disease.

cell biology↗

bioRxiv2020

MICROVASCULAR ENDOTHELIAL CELL ADAPTATION TO HYPOXIA IS ORGAN-SPECIFIC AND CONDITIONED BY ENVIRONMENTAL OXYGEN

SO_SCPLOWUMMARYC_SCPLOWMicrovascular endothelial cells (MVEC) are plastic, versatile and highly responsive cells, with morphological and functional aspects that uniquely match the tissues they supply. The response of these cells to oxygen oscillations is an essential aspect of tissue homeostasis, and is finely tuned to maintain organ function during physiological and metabolic challenges. Primary MVEC from two continuous capillary networks with distinct organ microenvironments, those of the lung and brain, were pre-conditioned at normal atmospheric ([~] 21 %) and physiological (5 and 10 %) O2 levels, and subsequently used to compare organ-specific MVEC hypoxia response. Brain MVEC preferentially stabilise HIF-2 in response to hypoxia, whereas lung MVEC primarily accumulate HIF-1; however, this does not result in significant differences at the level of transcriptional activation of hypoxia-induced genes. Glycolytic activity is comparable between brain and lung endothelial cells, and is affected by oxygen pre-conditioning, while glucose uptake is not changed by oxygen pre-conditioning and is observed to be consistently higher in brain MVEC. Conversely, MVEC mitochondrial activity is organ-specific; brain MVEC maintain a higher relative mitochondrial spare capacity at 5% O2, but not following hyperoxic priming. If maintained at supra-physiological O2 levels, both MVEC fail to respond to hypoxia, and have severely compromised and delayed induction of the glycolytic shifts required for survival, an effect which is particularly pronounced in brain MVEC. Oxygen preconditioning also differentially shapes the composition of the mitochondrial electron transport chain (ETC) in the two MVEC populations. Lung MVEC primed at physioxia have lower levels of all ETC complexes compared to hyperoxia, an effect exacerbated by hypoxia. Conversely, brain MVEC expanded in physioxia display increased complex II (SDH) activity, which is further augmented during hypoxia. SDH activity in brain MVEC primed at 21 % O2 is ablated; upon hypoxia, this results in the accumulation of near-toxic levels of succinate in these cells. Our data suggests that, even though MVEC are primarily glycolytic, mitochondrial integrity in brain MVEC is essential for metabolic responses to hypoxia; these responses are compromised when cells are exposed to supra-physiological levels of oxygen. This work demonstrates that the study of MVEC in normal cell culture environments do not adequately represent physiological parameters found in situ, and show that the unique metabolism and function of organ-specific MVEC can be reprogrammed by external oxygen, significantly affecting the timing and degree of downstream responses. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/265926v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@6a8668org.highwire.dtl.DTLVardef@19e3f17org.highwire.dtl.DTLVardef@3fffd5org.highwire.dtl.DTLVardef@1b517da_HPS_FORMAT_FIGEXP M_FIG C_FIG In briefHypoxia sensing by microvascular endothelial cells (MVEC) is organ-specific, and efficacy of response is affected by external oxygen. While glycolytic capacity is mostly maintained in MVEC regardless of organ or origin, mitochondrial function is required for adequate sensing and timely metabolic shift to glycolysis. Hyperoxygenation of MVEC compromises mitochondrial function, glycolytic shift and survival to hypoxia. HighlightsO_LIEnvironmental O2 influences MVEC hypoxia response in an organ-specific fashion C_LIO_LIBrain MVEC are unable to respond and survive to hypoxia if hyperoxygenated prior to stress C_LIO_LIMVEC glycolytic capacity is not affected by O2, but the increase in glucose uptake and shift to glycolytic metabolism stifled and delayed in hyperoxidized MVEC C_LIO_LIHigh O2 ablates activity of mitochondria complex II in brain MVEC, significantly disturbing succinate levels Disruption of mitochondrial integrity compromises hypoxia sensing irrespective of glycolytic capacity C_LI

cell biology↗

bioRxiv2020

Circulating levels of epirubicin cause endothelial senescence while compromising metabolic activity and vascular function

Anthracycline-based chemotherapy is a common treatment for cancer patients. Because it is delivered intravenously, endothelial cells are exposed first and to the highest concentrations, prior to diffusion to target cells. Not surprisingly, vascular dysfunction is a consequence of anthracycline therapy. While chemotherapy-induced endothelial damage at administration sites has been investigated, the effects of lower doses encountered by distant microvascular networks has not. The aim of this study was to investigate the impact of epirubicin, a widely used anthracycline, on healthy endothelial cells to elucidate its effects on microvascular physiology. Here, endothelial cells were briefly exposed to low doses of epirubicin to recapitulate levels in circulation following dilution in the blood and compound half-life in circulation. Both immediate and prolonged responses to treatment were assessed to determine changes in endothelial function. Epirubicin caused a decrease in proliferation and viability in hUVEC, with lower doses resulting in a senescent phenotype in a large proportion of cells, accompanied by a significant increase in pro-inflammatory cytokines and a significant decrease in metabolic activity. Epirubicin exposure also impaired endothelial function with delayed wound closure, reduced angiogenic potential and increased monolayer permeability downstream of VE-cadherin internalization. Primary lung endothelial cells obtained from epirubicin-treated mice similarly demonstrated reduced viability and functional impairment. In vivo, epirubicin treatment resulted in persistent reduction in lung vascular density and significantly increased infiltration of myeloid cells. Modulation of endothelial status and inflammatory tissue microenvironment observed in response to low doses of epirubicin may predict risk for long-term secondary pathologies associated with chemotherapy.

cell biology↗

bioRxiv2019

Fetus-derived IGF2 matches placental development to fetal demand

In all eutherian mammals, growth of the fetus is dependent upon a functional placenta, but whether and how the latter adapts to putative fetal signals is currently unknown. Here we demonstrate, through fetal, endothelial, hematopoietic and trophoblast-specific genetic manipulations in the mouse, that endothelial and fetus-derived IGF2 is required for the continuous expansion of the feto-placental microvasculature in late pregnancy. The effects of IGF2 on placental microvasculature expansion are mediated, in part, through IGF2R and angiopoietin-Tie2/TEK signalling. Additionally, IGF2 exerts IGF2R-ERK1/2-dependent pro-proliferative and angiogenic effects on primary feto-placental endothelial cells ex vivo. Endothelial and fetus-derived IGF2 also plays an important role in trophoblast morphogenesis, acting through Gcm1 and Synb. Thus, our study reveals a direct role for the imprinted Igf2-Igf2r axis on matching placental development to fetal growth and establishes the principle that hormone-like signals from the fetus play important roles in controlling placental microvasculature and trophoblast morphogenesis.

developmental biology↗