Detección de Células Cancerosas en Movimiento

Circulating tumor cells (CTCs) are those cells that detach from a primary or secondary tumor and spread to distant organs via the blood or lymph systems [1-5]. CTCs have been shown to correlate with metastatic disease state and their detection and quanti?cation may be used by clinicians to optimize the therapy of cancer patients. CTCs may occur as one cell among millions of normal blood cells and may originate from solid tumors or hematological malignancies such as leukemia. Many methods have been proposed to ?nd CTCs, but due to their rare occurrence no accepted method has been implemented in clinical practice. RT-PCR, immunomagnetic separation, micro-fluidics, and automated digital microscopy have all been investigated as means for CTC detection [6-8]. All of these methods suffer from a combination of sensitivity problem, the need for specialists for sample analysis, and slow processing time. RT-PCR in particular has been hampered by high cost and difficult preparation. While it has successfully been used to detect CTCs in a research setting, its peculiar absence in clinical diagnosis is due to these short comings. Automated digital microscopy has been used along with ?ber-optic array scanning, although the need for single cell passes requires several hours for testing a single sample. Immunomagnetic separation has been used widely in research and has even manifested as a commercial device, the CellSearch system. However, this system requires manual imaging of suspect cells and takes hours to process a single sample. Flow cytometry, however, is used routinely to count leukemia cells using ?uorescence detection [9]. This method uses high ?ow rates of single cells through a detection volume within which cells pass. This method is effective for ?nding large numbers of CTCs, although rare CTCs from solid tumors would probably pass unnoticed. For leukemia, CTCs are not rare events, as the disease itself is characterized by a proliferation of malignant cells in the blood stream. We have developed a system for detecting circulating melanomacells (CMCs) exploitingthenatural chromophore, melanin, that is present inmost melanoma cells (Fig. 1) [10]. It has been estimated that over 95% of all melanomas are pigmented, enabling the photoacoustic process to work [11,12]. Figure 1 shows the melanoma cell line we used in these experiments. Figure 1A,B shows melanin pigment directly, while immunofluorescence stains in Figure 1C,D show the presence of tyrosinase, a melanin precurser. In our method for CMC detection, we circulate a suspension of cells in a closed loop comprised of a peristaltic pump, silicone tubing, and a glass flow chamber. This glass flow chamber is irradiated with nanosecond-pulsed laser light, generating photoacoustic waves in suspended pigmented cells. Typically, this cell suspension would be the buffy coat separated from whole blood of ccer patients. The buffy coat is composed of white blood cells that have no inherent optical absorption. If the whole blood sample contained CMCs, they would separate with the buffy coat. Separation of the buffy coat is a standard practice and has been used in CTC research [13]. Due to density differences in blood components, centrifugation separates red blood cells, white blood cells, plasma, and lipids. CTCs have a similar density to white blood cells and therefore separate within the buffy coat. We used a Histopaque solution with a density of 1.077 g/ml to further demark the red blood cells from the buffy coat, allowing us to remove the buffy coat with a micropipette. We estimate from current work that we can recover 80-90% of melanoma cells spiked in whole blood after centrifugation. Thus, in the absence of pathological conditions that cause optical absorption in leukocytes, photoacoustic events generated in this flow system indicate the presence of melanoma cells in the circulatory system. Since the White blood cells have no absorption, we can pass thousands of cells through the active sensing area without compromising En este proyecto se esta construyendo un dispositivo óptico capaz de detectar células de melanoma in vitro e in vivo. Este dispositivo emplea el efecto PA para detectar y cuantificar células tumorales circulatorias, con la finalidad de detectar cáncer o como un método para verificar la efectividad un tratamiento de quimioterapia. Este método presenta la oportunidad de generar nuevos estándares protocolarios ya que permite subsanar muchas de las necesidades presentadas en los actuales métodos de diagnostico y tratamiento del cáncer. La principal hipótesis que subyace detrás del sistema de detección PA es la capacidad del efecto PA de identificar con precisión los estadios tempranos y tardíos en la proliferación de células cancerosas circulantes que se encuentran presente en el organismo que se estudie. Esto podrá agregar una alternativa indolora a los protocolos médicos que se basan en métodos quirúrgicos, así como también, proveer un mecanismo para la detección temprana de la enfermedad. Se ha propuesto que este sistema de detección pueda sustentar un método preciso y con un umbral de detección que no se había alcanzado con anterioridad alcanzando la identificación de células tumorales circulantes de manera individual que se encuentren en presencia de los diversos componentes de la sangre entera. Este proyecto, se esta realizando en colaboración con el Luis Polo-Parada de la Universidad de Missouri, EUA.

En este proyecto están realizado su tesis dos estudiantes, a saber

• Rafael Pérez Solano. Estudiante del Doctorado en Física. Su tema de investigación es Estudio teórico de la señal fotoacústica generada por sistemas micrométricos.
• Francisco Ignacio Ramírez Pérez. Estudiante de la Maestría en Fisica. Su tema de investigación es El estudio teórico-experimental de la senal fotoacústica generada por celulas de melanoma.