Prostate Cancer Heterogeneous High-Metastatic Multi-Organ-Colonizing Chemo-Resistant Variants Selected by Serial Metastatic Passage in Nude Mice Are Highly Enriched for Multinucleate Giant Cells

<div><p>In order to further understand the role of tumor heterogeneity in metastasis and chemo-resistance, high metastatic PC-3 human prostate cancer variants were selected by injecting parental PC-3 cells, expressing green fluorescent protein (GFP) in the footpad of nude mice, which then metastasize to inguinal lymph nodes. The PC-3-GFP cells which metastasized to the inguinal lymph nodes were collected and were re-injected to the footpad. After 6 such cycles, the PC-3-GFP cells collected from inguinal lymph nodes (PC-3-GFP-LN) were again injected to the footpad. PC-3-GFP-LN showed 100% metastasis to major lymph nodes (popliteal, inguinal, axillary, and cervical), and 100% metastasis to bone and lung. The percent of giant cell variants was enriched in PC-3-GFP-LN-6 compared to parental cells and increased with each cycle of selection, which in turn had increased metastasis. PC-3-GFP-LN-6 cells were resistant to 5-fluorouracil, doxorubicin and cisplatinum, compared to parental PC-3. However, PC-3-GFP-LN-6 was sensitive to the traditional Chinese medicine (TCM) herbal mixture LQ, similar to the parental cells. These results suggest that PC-3 tumors are heterogenous and that subpopulations of highly metastatic, drug-resistant cells can be step-wise selected using a mouse model of tumor progression.</p></div

Metastatic frequency.

<p>Metastasis frequency after in vivo metastasis selection cycles one to six of PC-3-GFP. After 6 cycles of selection, the PC-3-GFP-LN6 variant subline developed metastasis in the lung, bone, inguinal node, axillary node, and cervical node in all mice.</p

Sensitivity of the PC-3-GFP-LN3-6 cells to 5-fluorouracil (5-FU), cisplatinum (CDDP), doxorubicin (DOX), and traditional Chinese medicine herbal mixture LQ.

<p>PC-3-GFP-LN-6 was highly-resistant to all chemotherapeutic drugs tested compared to the parental PC-3-GFP cells (<i>p</i><0.01). PC-3-GFP-LN-6 only showed similar sensitivity to the parental cell line PC-3-GFP when treated with TCM LQ.</p

PC-3-GFP cells were injected to the footpad of mice.

<p>After 3 weeks, PC-3-GFP developed spontaneous metastasis in the popliteal lymph node and inguinal lymph node. The metastatic PC-3-GFP cells of the inguinal lymph node were collected and re-injected in the footpad to develop variants with increased metastatic potential. The cells in the inguinal node were collected and re-injected the footpad. After 6 such cycles of re-injection and selection of metastasis, the selected cell line developed 100% of metastasis in the lung, bone, inguinal node, axillary node, and cervical node (A&B). The morphology of in vitro cultured metastatic PC-3-GFP-LN cells, from each cycle, cultured from inguinal lymph node metastasis and parental PC-3-GFP are shown. The giant cell number was enriched with the cycle number, indicating that giant cells are more aggressive and highly metastatic cells.</p

Cellular morphology of PC-3-GFP-LN-6 (A-C).

<p>PC-3-GFP-LN-6 contained round, spindle and giant cells. The giant cells contain multiple nuclei (D-H). The number of nuclei per cell ranges between 2–22. Arrows show some of the multiple nuclei in a giant cell.</p

Determination of protein expression of lineage markers in cultured MDA-MB-435 cells.

<p><b>A</b>, immunofluorescence staining of epithelial (ESA), melanocytic (melan-A) and neuronal/glial markers (GFAP and nestin) in cultured MDA-MB-435, U87 glioblastoma and Ul-Mel melanoma cancer cell lines. <b>B</b>, immunohistochemistry staining of lineage markers in embedded MDA-MB-435 cells. AE1/AE3: pan-cytokeratin epithelial marker.</p

PCR analysis of mRNA expression of neuronal/glial, epithelial and melanocytic differentiation markers <i>in vitro</i>.

<p>Expression of epithelial (I), melanocytic (II) and neuronal/glial markers (III) by MCF-10A normal mammary epithelial cells (lane 1), breast cancer cell lines (lane 2–5), melanoma (lane 6), and glioblastoma cells (lane 7 and 8). TBP is used as a loading control.</p

Long-Term Extensive Ectopic Hair Growth on the Spinal Cord of Mice from Transplanted Whisker Follicles

<div><p>We have previously demonstrated that hair follicles contain nestin-expressing pluripotent stem cells that can effect nerve and spinal cord repair upon transplantation. In the present study, isolated whisker follicles from nestin-driven green fluorescent protein (ND-GFP) mice were histocultured on Gelfoam for 3 weeks for the purpose of transplantation to the spinal cord to heal an induced injury. The hair shaft was cut off from Gelfoam-histocultured whisker follicles, and the remaining part of the whisker follicles containing GFP-nestin expressing pluripotent stem cells were transplanted into the injured spinal cord of nude mice, along with the Gelfoam. After 90 days, the mice were sacrificed and the spinal cord lesion was observed to have healed. ND-GFP expression was intense at the healed area of the spinal cord, as observed by fluorescence microscopy, demonstrating that the hair follicle stem cells were involved in healing the spinal cord. Unexpectedly, the transplanted whisker follicles sprouted out remarkably long hair shafts in the spinal cord during the 90 days after transplantation of Gelfoam whisker histocultures to the injured spine. The pigmented hair fibers, grown from the transplanted whisker histocultures, curved and enclosed the spinal cord. The unanticipated results demonstrate the great potential of hair growth after transplantation of Gelfoam hair follicle histocultures, even at an ectopic site.</p></div

MDA-MB-435 lung cell lines derived from lung metastases display neuronal/glial differentiation morphology.

<p><b>A</b> and <b>E</b>, parental MDA-MB-435-GFP cells. <b>B–D</b> and <b>F–H</b>, three representative MDA-MB-435-GFP-L cell lines exhibit morphological features of well-differentiated neuron/glial cells.</p

Determination of protein expression of lineage markers in MDA-MB-435 xenografts and in metastatic lesions.

<p>Immunohistochemical detection of epithelial (AE1/AE3, ESA), melanocytic (melan-A), and neuronal/glial markers (GFAP, nestin) in orthotopic mammary fat pad tumors, and mammary duct differentiation (H&E) (<b>A</b>), pleural lung macrometastases (<b>B</b>) and lung micrometastases (<b>C</b>, arrows).</p