Fluorescence monitoring of rare circulating tumor cell and cluster dissemination in a multiple myeloma xenograft model in vivo

Abstract

Circulating tumor cells (CTCs) are of great interest in cancer research because of their crucial role in hematogenous metastasis. We recently developed “diffuse in vivo flow cytometry” (DiFC), a preclinical research tool for enumerating extremely rare fluorescently labeled CTCs directly in vivo. In this work, we developed a green fluorescent protein (GFP)-compatible version of DiFC and used it to noninvasively monitor tumor cell numbers in circulation in a multiple myeloma (MM) disseminated xenograft mouse model. We show that DiFC allowed enumeration of CTCs in individual mice overtime during MM growth, with sensitivity below 1 CTC mL − 1 of peripheral blood. DiFC also revealed the presence of CTC clusters (CTCCs) in circulation to our knowledge for the first time in this model and allowed us to calculate CTCC size, frequency, and kinetics of shedding. We anticipate that the unique capabilities of DiFC will have many uses in preclinical study of metastasis, in particular, with a large number of GFP-expressing xenograft and transgenic mouse models.

Keywords: circulating tumor cell clusters; circulating tumor cells; fluorescence; multiple myeloma; optical devices.

Fig. 1

(a) GFP-DiFC instrument schematic and fiber probe design (inset; see text for details). (b) The DiFC system was mounted on an optics cart that could be moved easily between sites. (c) The fiber bundles were placed on the ventral surface of the mouse tail approximately above the ventral caudal bundle. (d) DiFC allows detection and discrimination of circulating MM GFP-labeled CTCs moving the arterial and venous directions. Peaks that were not measured in both channels were assumed to be moving in smaller blood vessels or due to noise, and were subsequently discarded from the analysis.

Fig. 2

The FC gating strategy for counting MM.1S.GFP.Luc cells is shown. (a) SSC-FSC plot for MM.1S cells in culture. (b) The blue (BL1) fluorescence of MM.1S.GFP.Luc cells and DG2 microspheres. The mode intensity of DG2 microspheres was used as counting threshold since it was lower than cultured MM.1S.GFP.Luc cells. (c) SSC-FSC for a blood sample drawn from a mouse, with gate shown. RBCs were first depleted using a lysate. (d) Blue (GFP) fluorescence histogram of cells in blood. Most peaks are low-fluorescence debris or unlabeled cells. (e) Blue (GFP) fluorescence histogram of cells exceeding the DG2 threshold only, yielding the cell counts in the sample.

Fig. 3

Validation of the GFP-DiFC system in (a) a flow phantom model with (b) PBS or (c) DG4 fluorescent microspheres. (d) We also verified that the GFP-DiFC system could detect MM.1s labeled cells in vivo. Data from (e) control and (f) MM.1s-injected mice are shown. See text for details.

Fig. 4

We performed BLI imaging weekly for all mice for 1 month following tail vein injection of MM.1S.GFP.Luc cells. (a)–(c) Luminescence increased as the MM disease grew in the bone marrow, which was first observable in small areas of (b) the spine by day 23. (c) By day 30, diffuse patterns of MM growth were observable in the skull, spine, and hips. (d) This general pattern was observed for all inoculated mice except for one (M3C2), with significant interexperimental variability between mice.

Fig. 5

We performed DiFC scanning twice per week during the development of the xenograft model. (a) An example 10-min DiFC sequence from a control (PBS injected) mouse is shown, illustrative of the low FAR of the DiFC system. (b)–(e) As the MM disease progressed, GFP+ MM cells were observable in circulation with increasing frequency. (f) The mean single-cell count rate in the arterial direction for all mice is shown, showing growth over the course of the disease, as well as significant interexperimental variability. (g) An expanded view of (f) for mean DiFC-count rates between 0 and $1\mathrm{counts}{\min }^{-1}$. DiFC allowed detection of very low numbers of circulating cells above the FAR (dotted line, $0.016{\min }^{-1}$). As with BLI, one mouse (M3C2) failed to show signs of MM growth. (h) A transient increase in DiFC count was frequently observed in the first 5 to 10 min of scanning, which we attribute to warming of the mouse tail and corresponding increase in blood flow.

Fig. 6

(a) Raster plot showing the detection times of arterial-matched CTCs for mouse M2C1 on day 31 during DiFC scanning, where each vertical line represents one detected cell. (b) The average count rate for this scan was $7.7{\min }^{-1}$ (red line), but significant variability was observed when considering a 60-s moving average window (blue dotted line).

Fig. 7

(a)–(d) DiFC also showed large, irregularly shaped signals (relative to individual cells), which we hypothesized were due to the presence of CTCCs. (e) We used a threshold of 300-nA amplitude and 100-ms peak width to identify CTCCs. (f) These were observed with MM-injected mice only (never controls) and appeared approximately as soon as single CTCs were observed in circulation at a rate of $\sim 10\%$ of the arterial count rate. (g) Analysis of the peak amplitudes allowed us to estimate the sizes of the clusters, which were frequently fewer than 10 cells, but individual (inset) clusters were very large, with dozens of cells. (h)–(s) Example white-light, GFP fluorescence, and merged (overlay) blood smear micrographs from MM.1S bearing mice sacrificed on day 36 after injection. Most observed MM $\mathrm{GFP}+$ cells were single cells (h)–(j), but small numbers of CTCCs (k)–(s) were also observed as predicted from our DiFC measurements. Each image is $65\times 65{\mu \mathrm{m}}^2$.

Fig. 8

(a) We calculated the “corrected” DiFC count rate, which combined contributions from single CTCs and CTCCs (see text for details). (b) We drew blood samples on days 24 and 31 and counted MM GFP+ cells in the blood. (c) We compared these to the DiFC count rate on the same days, allowing us to estimate the detection sensitivity of DiFC. (d) The DiFC count rate also linearly correlated with MM disease burden measured by BLI for all mice and time-points ($r^2=0.75$). All indicated points are above the FAR of the system (black dotted line).