RESUMO
Flow-based cytometry methods are widely used to analyze heterogeneous cell populations. However, their use for small molecule studies remains limited due to bulky fluorescent labels that often interfere with biochemical activity in cells. In contrast, radiotracers require minimal modification of their target molecules and can track biochemical processes with negligible interference and high specificity. Here, we introduce flow radiocytometry (FRCM) that broadens the scope of current cytometry methods to include beta-emitting radiotracers as probes for single cell studies. FRCM uses droplet microfluidics and radiofluorogenesis to translate the radioactivity of single cells into a fluorescent signal that is then read out using a high-throughput optofluidic device. As a proof of concept, we quantitated [18F]fluorodeoxyglucose radiotracer uptake in single human breast cancer cells and successfully assessed the metabolic flux of glucose and its heterogeneity at the cellular level. We believe FRCM has potential applications ranging from analytical assays for cancer and other diseases to development of small-molecule drugs.
Assuntos
Técnicas Biossensoriais , Bioensaio , Citometria de Fluxo , Humanos , Microfluídica , Fenômenos FísicosRESUMO
Hypoxia-inducible factor 1 (HIF-1) is a master transcription factor that controls cellular homeostasis. Although its activation benefits normal tissue, HIF-1 activation in tumors is a major risk factor for angiogenesis, therapeutic resistance, and poor prognosis. HIF-1 activity is usually suppressed under normoxic conditions because of rapid oxygen-dependent degradation of HIF-1α. Here, we show that, under normoxic conditions, HIF-1α is upregulated in tumor cells in response to doxorubicin, a chemotherapeutic agent used to treat many cancers. In addition, doxorubicin enhanced VEGF secretion by normoxic tumor cells and stimulated tumor angiogenesis. Doxorubicin-induced accumulation of HIF-1α in normoxic cells was caused by increased expression and activation of STAT1, the activation of which stimulated expression of iNOS and its synthesis of nitric oxide (NO) in tumor cells. Mechanistic investigations established that blocking NO synthesis or STAT1 activation was sufficient to attenuate the HIF-1α accumulation induced by doxorubicin in normoxic cancer cells. To our knowledge, this is the first report that a chemotherapeutic drug can induce HIF-1α accumulation in normoxic cells, an efficacy-limiting activity. Our results argue that HIF-1α-targeting strategies may enhance doxorubicin efficacy. More generally, they suggest a broader perspective on the design of combination chemotherapy approaches with immediate clinical impact.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/metabolismo , Doxorrubicina/farmacologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Fatores de Transcrição/genética , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Células MCF-7 , Neoplasias Mamárias Experimentais/tratamento farmacológico , Neoplasias Mamárias Experimentais/genética , Neoplasias Mamárias Experimentais/metabolismo , Camundongos , Camundongos Nus , Óxido Nítrico/genética , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Distribuição Aleatória , Fator de Transcrição STAT1/deficiência , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/metabolismo , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
Resistance of hypoxic solid tumor niches to chemotherapy and radiotherapy remains a major scientific challenge that calls for conceptually new approaches. Here we exploit a hitherto unrecognized ability of sickled erythrocytes (SSRBCs) but not normal RBCs (NLRBCs) to selectively target hypoxic tumor vascular microenviroment and induce diffuse vaso-occlusion. Within minutes after injection SSRBCs, but not NLRBCs, home and adhere to hypoxic 4T1 tumor vasculature with hemoglobin saturation levels at or below 10% that are distributed over 70% of the tumor space. The bound SSRBCs thereupon form microaggregates that obstruct/occlude up to 88% of tumor microvessels. Importantly, SSRBCs, but not normal RBCs, combined with exogenous prooxidant zinc protoporphyrin (ZnPP) induce a potent tumoricidal response via a mutual potentiating mechanism. In a clonogenic tumor cell survival assay, SSRBC surrogate hemin, along with H(2)O(2) and ZnPP demonstrate a similar mutual potentiation and tumoricidal effect. In contrast to existing treatments directed only to the hypoxic tumor cell, the present approach targets the hypoxic tumor vascular environment and induces injury to both tumor microvessels and tumor cells using intrinsic SSRBC-derived oxidants and locally generated ROS. Thus, the SSRBC appears to be a potent new tool for treatment of hypoxic solid tumors, which are notable for their resistance to existing cancer treatments.