Differences in absorption and/or scattering of cancerous and normal skin have the potential to provide a basis for noninvasive cancer detection. In this study, we have determined and compared the in vitro optical properties of human epidermis, dermis, and subcutaneous fat with those of nonmelanoma skin cancers in the spectral range from 370 to 1600 nm. Fresh specimens of normal and cancerous human skin were obtained from surgeries. The samples were rinsed in saline solution and sectioned. Diffuse reflectance and total transmittance were measured using an integrating sphere spectrophotometer. Absorption and reduced scattering coefficients were calculated from the measured quantities using an inverse Monte Carlo technique. The differences between optical properties of each normal tissue-cancer pair were statistically analyzed. The results indicate that there are significant differences in the scattering of cancerous and healthy tissues in the spectral range from 1050 to 1400 nm. In this spectral region, the scattering of cancerous lesions is consistently lower than that of normal tissues, whereas absorption does not differ significantly, with the exception of nodular basal cell carcinomas (BCC). Nodular BCCs exhibit significantly lower absorption as compared to normal skin. Therefore, the spectral range between 1050 and 1400 nm appears to be optimal for nonmelanoma skin cancer detection.

An in vivo flow cytometer is developed that allows the real-time detection and quantification of circulating fluorescently labeled cells in live animals. A signal from a cell population of interest is recorded as the cells pass through a slit of light focused across a blood vessel. Confocal detection of the excited fluorescence allows continuous monitoring of labeled cells in the upper layers of scattering tissue, such as the skin. The device is used to characterize the in vivo kinetics of red and white blood cells circulating in the vasculatúre of the mouse ear. Potential applications in biology and medicine are discussed.Current methods to detect and quantify various types of cells within the blood stream involve extraction of blood from the patient or animal followed by ex vivo labeling and detection. For example, standard flow cytometry involves taking blood samples, fluorescently labeling specific cell populations, and passing these cells in a single file through a flow stream. 1 The cells are interrogated by a light source (usually a laser) to determine the types and number of cells based on their fluorescence and light-scattering signals. Another example is a hemocytometer, which involves manual counting of cells against a grid while viewing them with a microscope. Although both methods are useful, they provide only a single time sample. Consequently, if the cell population of interest varies unpredictably or rapidly with time, it is difficult to obtain a valid temporal population profile, since it is difficult to know when to sample. In addition, with both methods, blood must be withdrawn for each time point, and there is a significant time delay between blood withdrawal and analysis. The development of confocal and two-photon imaging techniques has allowed the detection of static and circulating fluorescently labeled cells in vivo. 2 However, extraction of quantitative information about the number and flow characteristics of a specific cell population can be extremely tedious. In addition, the high velocity of flowing cells, especially in the arterial circulation, makes it difficult and sometimes impossible to track the cells, even when images are captured at video rates. To remedy these problems, we have constructed a flow cytometer with the capability of detecting and quantifying the number and flow characteristics of fluorescently labeled cells in vivo and over a continuous time period.The underlying principle of operation of the in vivo flow cytometer is confocal excitation and detection of fluorescently labeled cells in circulation. A schematic of the experimental setup is shown in Fig. 1. The animal to be studied is anesthetized and placed on the stage with its ear adhered to a microscope slide with glycerine. A blood vessel of appropriate diameter is identified (see below). Light from a He-Ne laser is then focused into a slit by a cylindrical lens and imaged across the selected blood vessel with a microscope objective lens (40×, 0.6 NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA...

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The fate of circulating tumor cells is an important determinant of their ability to form distant metastasis. Here, we demonstrate the use of in vivo flow cytometry as a powerful new method for detecting quantitatively circulating cancer cells. We specifically examine the circulation kinetics of two prostate cancer cell lines with different metastatic potential in mice and rats. We find that the cell line and the host environment affect the circulation kinetics of prostate cancer cells, with the intrinsic cell line properties determining the initial rate of cell depletion from the circulation and the host affecting cell circulation at later time points.

Survival of cancer cells in the circulation is an important step in metastasis. However, the fate of circulating tumor cells is difficult to assess with conventional methods that require blood sampling. We report the first in situ measurement of circulating apoptotic cells in live animals using in vivo flow cytometry, a novel method [1-3] that enables real-time detection and quantification of circulating cells without blood extraction. Injected cancer cells undergo cell death within 1 -2 hr after entering the mouse circulation. Apoptotic cells are rapidly cleared from the circulation with a half-life of~10 min. Real-time monitoring of circulating apoptotic cells can be useful for detecting early changes in disease processes, as well as for monitoring response to therapeutic intervention.To detect circulating apoptotic cells in vivo, annexin-V conjugated to Alexa Fluor 647 (AF647) was used to label exposed phosphatidylserine on the cell surface. The long wavelength of AF647 fluorescence (Molecular Probes, Eugene, OR) allows its detection through blood with minimum attenuation by red blood cells. In initial studies conducted to demonstrate that our instrument had sufficient sensitivity to detect individual annexin-V-labeled apoptotic cells in vivo, we used MatLyLu prostate cancer cells pretreated with camptothecin [4] as a positive control. We verified that >80% of the camptothecin-treated cells undergo apoptosis by conventional flow cytometry (i.e., >80% of the treated cells were FITC -annexin V positive and propidium iodide negative). The camptothecin-treated cells were then labeled with the AF647-conjugated annexin-V and injected into the mouse intravenously. The circulating annexin-V + cells were measured by focusing a He -Ne laser beam onto an ear vessel and detecting the fluorescent bursts as individual cells flowed through the probe beam (Figure 1, inset). The cell count dropped from >200/min to <50/min within 20 min of injection, indicating rapid clearance (Figure 1, solid circles).Next, unlabeled camptothecin-treated apoptotic Mat-LyLu cells were introduced into the circulation, followed immediately by injection of the annexin-V probe. The cell count increased during the first 5 min (Figure 1, open squares) due to the in vivo labeling process. Subsequently, the cells were cleared with the same rate as with in vitro labeled cells. Control experiments with injection of annexin-V alone yielded negligible cell count (Figure 1, crosses).When untreated, viable MatLyLu tumor cells were injected into the circulation; no apoptotic cells were detected initially. Circulating annexin-V + cells were detected starting at 1 hr and reached a plateau at 3 hr (Figure 2, solid circles). Because the total number of circulating MatLyLu cells decreased precipitously during the first 3 hours [2], we conclude that the fraction of MatLyLu cells undergoing cell death steadily increases during this time. Analysis of extracted blood with standard flow cytometry indicated that about 9% of the injected cells were annexin-V + at the 3-h...

The in vivo flow cytometer enables the real-time detection and quantification of fluorescent cells circulating within a live animal without the need for incisions or extraction of blood. It has been used in demonstrating flow velocity disparities in biological flows, and in the investigation of the circulation kinetics of various types of cells. However, a shortcoming of this in vivo flow cytometer is that it provides only one excitation slit at one wavelength, resulting in several performance limitations. Therefore, a second in vivo flow cytometer that provides two different laser wavelengths, 473 and 633 nm, and one or two excitation slits has been designed and built. Thus far, the two-color system has been used to acquire circulation kinetics data of two different cell populations each labeled with a different marker, one cell population labeled with two different markers, and one cell population expressing the green-fluorescent protein gene. In addition, accurate arterial red blood cell velocities within a mouse have been determined using the cytometer.

Human granulocytes (polymorphonuclear leukocytes, PMN) produce H2O2 and other reactive oxygen species while undergoing phagocytosis. To examine the role of the glutathione cycle in metabolizing H2O2, we incubated PMN with 1,3-bis (2-chloroethyl) nitrosourea (BCNU). Incubation of PMN with BCNU results in a dose-dependent inhibition of PMN glutathione reductase (GRED), with 50% inhibition occurring at approximately 2 micrograms/mL BCNU. PMN hexose monophosphate shunt activity stimulated with an exogenous H2O2-generating system was inhibited only when the GRED activity was reduced to less than 30% of control. BCNU-treated cells contained lower levels of reduced sulfhydryls and reduced glutathione, which decreased even more in the presence of an exogenous H2O2-generating system. The effect of BCNU and exogenous H2O2 on various aspects of phagocytosis were examined. Exposure of BCNU-treated PMN to an H2O2-generating system resulted in an inhibition of chemotactic peptide-induced shape changes and degranulation. The ability of BCNU-treated cells to produce O2- was diminished only when the PMN were incubated with an H2O2-generating system in the presence of cyanide. Ingestion of opsonized bacteria by BCNU-treated PMN was unaffected by incubation in an H2O2-generating system even in the presence of cyanide. We conclude that PMN GRED is inhibited by BCNU, the ability of PMN to metabolize H2O2 is affected only when GRED is reduced more than 70%, this inhibition affects the glutathione content of these cells, and some, but not all of the phagocytic functions of GRED-inhibited PMN are inhibited after exposure to an H2O2-generating system.

A simple technique has been used experimentally to produce in vitro Chinese hamster ovary cells with growth fractions ranging from 0 to 100%. Known numbers of exponentially growing and plateau‐phase tissue culture cells were mixed in various proportions to yield the desired final growth fraction. Cells attach to the culture flask surface within 1 hr of mixing. Treatment at that time with the nitrosourea compounds, CCNU and MeCCNU, resulted in differential drug survival sensitivities that were dependent upon the growth fraction of the population treated.