Expression of putative stem cell markers in Clone 66 murine breast cancer cells.

<p>(<b>A</b>) After staining Cl66 cells with Hoechst 33342 dye followed by FACS analysis, we detected 1.23% ±0.95% cells were SP cells (n = 3). (<b>B</b>) When Cl66 cells were FACS analyzed after Aldefluor treatment with or without DEAB (ALDH1 inhibitor), we found approximately 4.16% ±3.26% (n = 3) cells were ALDH1⁺. (<b>C</b>) After staining with anti-CD34 antibody followed by FACS analysis, we identified 90% ±13%, n = 4 cells were positive for CD34. Cells expressed highest levels of CD34 (CD34^high) and lowest levels of CD34 (CD34^low) were selected and sorted for this study. (<b>D</b>) After staining with anti-CD49f antibody followed by FACS analysis, we identified 99.47% ±0.21% (n = 3) cells were positive for CD49f. Cells with the highest levels of CD34 expression (CD34^high) and lowest levels of CD34 expression (CD34^low) were selected and sorted for this study. For the SP analysis, an X-cite LightWave air-cooled 20 mW UV laser at 354 nm (made by JDS Uniphase) was used. For the ALDH1, PE, FITC and Alexa488 analysis, a Saphire air-cooled 100 mW blue laser at 488 nm (made by Coherent) was used. Average data from more than two independent assays are shown. n = number(s) of assay(s) performed.</p

Cell populations with stem phenotypes exhibited reduced proliferation and lower colony formation.

<p>(<b>A</b>) In proliferation analyses, when the number of cells was plotted against the incubation period (days), the proliferation rate was much higher in ALDH1⁺, non-SP (non-side population), CD34^low, and CD49f^high than their respective counterparts. Matrigel™ only favored the proliferation of the CD49f^low/neg population. Population doubling times of ALDH1⁺and CD49f^high cells (17±0.2, 20±0.4 hours) was less than ALDH1⁻ and CD49f^low cells (21±0.26, 24±0.41 hours) when calculated from the number of cells growing in log phase (day 2 to 6) using the formula, T_d⁼0.693 t/ln (N_t/N₀). CD34^high showed higher (25±0.43 hours) population doubling time than CD34^low (20±0.33 hours). With Matrigel™ indicated by +M. (<b>B</b>) When analyzed for colony formation efficiencies, cell populations with ALDH1⁻, non-SP, CD34^high, CD49f^high phenotypes showed higher colony forming efficiency than their respective counterparts. The CD49f^high and CD49f^low/neg cells did not show major differences in colony forming efficiencies, suggesting that this phenotype might not be a suitable phenotype to differentiate stem and non-stem phenotype, at least under <i>in vitro</i> culture conditions. Matrigel™ increased colony formation only for the CD34^low population. With Matrigel™ indicated by +M.</p

Histological analyses of tumors generated in mammary fat pads of Balb/c mice.

<p>Tumors were generated from SP, non-SP, ALDH1⁺, or ALDH1⁻ cells transplanted orthotopically in mammary fat pads of Balb/c mice. (<b>A top panel</b>) H & E stained tumor sections generated from SP and non-SP cells demonstrated the presence of blood vessels, inflammatory/immune cells with heterochromatic nuclei, and cells with secretion vesicles. Metaphase cells were only evident in SP tumor sections. (<b>A bottom panel</b>) H & E stained tumor sections generated from ALDH1⁺ and ALDH1⁻ demonstrated the presence of blood vessels, inflammatory/immune cells with heterochromatic nuclei, and cells with secretion vesicles. Metaphase cells were evident in ALDH1⁻ tumor sections. (<b>B</b>) non-SP cell derived tumors showed higher numbers of blood vessels (approx. 13) than SP cell derived tumors (approx. 3) and ALDH1⁻ cell derived tumors exhibited more (approx. 7) blood vessels than ALDH1⁺ derived tumors. (approx. 4). (<b>C</b>) Histological analyses of tumor sections generated from orthotopically re-transplanted pooled populations of SP, and non-SP derived tumor cells. Sections demonstrated the presence of blood vessels, inflammatory/immune cells with heterochromatic nuclei and secretion vesicles however, sections of re-transplanted pooled SP population derived tumor cells exhibited more blood vessels (≥3) than purified SP population derived tumors (≤3) and re-transplanted pooled non-SP population tumors exhibited fewer blood vessels (5–6) than purified non-SP population derived tumors (9–13). Cells in metaphase were only observed in re-transplanted pooled SP cell derived tumors. Original magnification, 400×. * = blood vessels; I/I = Inflammatory/immune cells with heterochromatic nuclei; SV = secretion vesicles; M = Cell in metaphase.</p

Levels and numbers of cytokines/chemokines produced by Cl66 alone and co-cultured Cl66 with MS-5 stromal cells.

<p>Media were collected at day 3 from Cl66 cells alone and co-cultured Cl66 with MS-5 stromal cells; and subjected to analysis of the cytokine/chemokine profiles. (<b>A</b>) Histograms show the production of chemokine(s)/cytokine(s) by Cl66 cells alone and changes influenced by co-culture with MS-5 stromal cells. (<b>B</b>) Histograms demonstrate the production of chemokine(s)/cytokine(s) during <i>in vitro</i> interactions of Cl66 with MS-5 stromal cells.</p

Various cell populations (%) with stem cell phenotypes were evaluated in Clone 66.

<p>Various cell populations (%) with stem cell phenotypes were evaluated in Clone 66.</p

SP cell populations with stem phenotypes have higher tumorigenic potentials.

<p>Various cell populations with stem phenotypes were transplanted orthotopically at different dilutions (24000, 10000, 6000, 3000, 1000, 500, 200, 50, or 25 cells/fat pad) with 1∶1 Matrigel™ into mouse mammary fat pads. Tumor volumes were calculated every alternate day for 6 weeks. When tumor growth was below 10 mm³, mice were necropsied following guideline and tumors were excised and analyzed. (<b>A</b>) Results indicated that fewer SP cells were needed to form tumors than ALDH1⁺cells, suggesting heterogeneities in tumorigenicity. A characteristic that might be related to these heterogeneities was levels of cytokines/chemokines produced by the differing cell populations. (<b>B</b>) ALDH1⁺ and ALDH1⁻ did not show consistent differences in tumor formation. (<b>C</b>) Volumes of tumors formed from 6000 cells of SP, non-SP, ALDH1⁺ and ALDH1⁻ cell populations showed that SP grew faster than non-SP, ALDH1⁺ and ALDH1⁻ cells.</p

Cell populations with stem phenotypes exhibited enhanced agar-colony formation.

<p>Agar colonies were obtained by culture of sorted cells from mouse mammary carcinoma Cl66 cells cultured in soft-agar containing complete DMEM media. Stem-type cells formed higher numbers of agar colonies than non-stem type cells. ALDH1⁺ and ALDH1⁻ formed more agar colonies than SP cells and SP cells formed more agar colonies than non-SP cells. Soft agar colony formation was much lower in CD34^high and CD49f^high cell populations. Matrigel™ increased the size of agar colonies of all cell populations, indicating that all cell populations responded to the growth factors present in Matrigel™. With Matrigel™ indicated by +M. All data presented are the average of 2 independent experiments.</p

Visulization 1.mp4

Visualization 1. Respiratory motion corrected time course images of ICG biodistribution in whole SS and SS.BN3 rats

Vascular Interventional Radiology-Guided Photothermal Therapy of Colorectal Cancer Liver Metastasis with Theranostic Gold Nanorods

We report sub-100 nm optical/magnetic resonance (MR)/X-ray contrast-bearing theranostic nanoparticles (TNPs) for interventional image-guided photothermal therapy (PTT) of solid tumors. TNPs were composed of Au@Gd₂O₃:Ln (Ln = Yb/Er) with X-ray contrast (∼486 HU; 10¹⁴ NPs/mL, 0.167 nM) and MR contrast (∼1.1 × 10⁸ mM^–1 S^–1 at 9.4 T field strength). Although TNPs are deposited in tumors following systemic administration <i>via</i> enhanced permeation and retention effect, the delivered dose to tumors is typically low; this can adversely impact the efficacy of PTT. To overcome this limitation, we investigated the feasibility of site-selective hepatic image-guided delivery of TNPs in rats bearing colorectal liver metastasis (CRLM). The mesenteric vein of tumor-bearing rats was catheterized, and TNPs were infused into the liver by accessing the portal vein for site-selective delivery. The uptake of TNPs with hepatic delivery was compared with systemic administration. MR imaging confirmed that delivery <i>via</i> the hepatic portal vein can double the CRLM tumor-to-liver contrast compared with systemic administration. Photothermal ablation was performed by inserting a 100 μm fiber-optic carrying 808 nm light <i>via</i> a JB1, 3-French catheter for 3 min under DynaCT image guidance. Histological analysis revealed that the thermal damage was largely confined to the tumor region with minimal damage to the adjacent liver tissue. Transmission electron microscopy imaging validated the stability of core–shell structure of TNPs <i>in vivo</i> pre- and post-PTT. TNPs comprising Gd-shell-coated Au nanorods can be effectively employed for the site-directed PTT of CRLM by leveraging interventional radiology methods

sj-docx-1-tct-10.1177_15330338231189593 - Supplemental material for X-ray and MR Contrast Bearing Nanoparticles Enhance the Therapeutic Response of Image-Guided Radiation Therapy for Oral Cancer

Supplemental material, sj-docx-1-tct-10.1177_15330338231189593 for X-ray and MR Contrast Bearing Nanoparticles Enhance the Therapeutic Response of Image-Guided Radiation Therapy for Oral Cancer by Gayatri Sharma, Mir Hadi Razeghi Kondelaji and Guru P. Sharma, Christopher Hansen, Abdul K. Parchur, Shayan Shafiee, Jaidip M. Jagtap, Brian Fish, Carmen Bergom, Eric Paulson, William A. Hall, Heather A. Himburg, Amit Joshi in Technology in Cancer Research & Treatment</p