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

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

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

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

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

Toxicity of treatment on the renal cortex.

<p>(A) Renal cortex of untreated mouse. (B) The renal cortex of mice treated with CTX demonstrated renal toxicity. Toxicity was mainly in the proximal tubular cells, including degeneration, obstruction, necrosis and swelling. (C) The renal cortex of mice treated with LQ had no toxicity.</p

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Multiple myeloma (MM) is the second most prevalent hematologic malignancy which remains uncurable. Numerous drugs have been discovered to inhibit MM cells. Indisulam, an aryl sulfonamide, has a potent anti-myeloma activity in vitro and in vivo. This study aims to explore the new mechanism of indisulam and investigate its potential use in combination with melphalan. We examined DNA damage in MM cells through various methods such as western blotting (WB), immunofluorescence, and comet assay. We also identified the role of topoisomerase IIα (TOP2A) using bioinformatic analyses. The impact of indisulam on the RNA and protein levels of TOP2A was investigated through qPCR and WB. Cell proliferation and apoptosis were assessed using CCK-8 assays, Annexin V/PI assays and WB. We predicted the synergistic effect of the combination treatment based on calculations performed on a website, and further explored the effect of indisulam in combination with melphalan on MM cell lines and xenografts. RNA sequencing data and basic experiments indicated that indisulam caused DNA damage and inhibited TOP2A expression by decreasing transcription and promoting degradation via the proteasome pathway. Functional experiments revealed that silencing TOP2A inhibited cell proliferation and induced apoptosis and DNA damage. Finally, Indisulam/melphalan combination treatment demonstrated a strong synergistic anti-tumor effect compared to single-agent treatments in vitro and in vivo. These findings suggest that combination therapies incorporating indisulam and melphalan have the potential to enhance treatment outcomes for MM.</div

Efficacy of LQ on experimental lung metastasis.

<p>Six mice were used in each group. Individual images of excised lungs from the mice in the experimental metastasis model were obtained. LLC-RFP cancer cells (2×10⁶) were injected into the tail vein of nude mice or C57BL/6 mice. From day 2, the mice were given PBS (po) every day as the untreated control. Mice were treated with LQ (600 mg/kg/day po) for 10 days. The LQ-treated mice had significantly reduced lung weight (<i>p<</i>0.001) and reduced red fluorescence area in the lung (<i>p<</i>0.001) compared to the untreated PBS controls. <b>A</b> and <b>B</b> are images of the lungs. <b>C–D</b> are lung weights. <b>E</b> is the total red fluorescence area.</p

Efficacy of LQ on tumor size, growth and weight in subcutaneous nude mouse tumor models.

<p>For the H460 and A549 cell lines, each treatment group contained 6 mice and 10 mice were used for the untreated control groups. For the LLC cell line, each treatment group contained 8 mice and 16 mice were used for the untreated control group. After the subcutaneous tumors grew, the nude mice were given either CTX (30 mg/kg/day i.p.) for 7 days or PX (600 mg/kg/day p.o) for 10 days. PBS (po) was used in the control group. Mice were treated at 150, 300, and 600 mg/kg/day of LQ (po) for 10 days. Tumor size was measured twice a week. Tumor weight was measured at the endpoint. Statistical significance between groups was determined with the Student’s <i>t</i>-test. H460 (<b>A&D</b>), A549 (<b>B&E</b>), and LLC (<b>C&F</b>) had growth inhibition and tumor size inhibition after treatment with all agents. The tumor weight was also significantly inhibited by all agents (<b>G</b>) (<i>p<</i>0.01). After LQ treatment, lung tumor growth and tumor weight were significantly inhibited, compared to the untreated control group in all models (<i>p<</i>0.01). CTX and PX treatment resulted in a significant inhibition of tumor weight (<i>P<</i>0.01) compared to the control group for all cell lines. LQ had more efficacy than PX (<i>p<</i>0.05) on all cell lines (<b>G</b>). CTX induced loss of body weight (<i>p<</i>0.05), but not LQ or PX (<b>H</b>).</p

Effect of LQ on tumor blood vessels.

<p>LLC-RFP cells were grown in nestin-driven GFP (ND-GFP) transgenic nude mice in which nascent blood vessel expressed GFP. Three mice were used in each group. In the LQ-treated mice, tumor blood vessels appeared to be destroyed. The GFP florescence of blood vessels was quantitated for area, density, integrated optical density (IOD), length and counts. The measurements indicated that LQ decreased blood vessels by 50%.</p