breast cancer cell growth  (Bioscientifica Ltd)

Journal: Journal of Nuclear Medicine

Article Title: Preirradiation of Spheroids with 225 Ac-Trastuzumab Improves Penetration of 225 Ac-Liposomes and MIRDcell Predictions of Responses to Drug Cocktails

doi: 10.2967/jnumed.124.269273

Figure Lengend Snippet: Penetration data in BT474 spheroids (200-µm radius) for Alexa Fluor 647-NHS-trastuzumab ( ), CFDA-SE liposomes ( ), and CFDA-SE liposomes after preirradiating spheroids with 6.5 kBq/mL 225 Ac-trastuzumab ( ). Shown are time-integrated average concentrations as function of distance from centers of spheroids (A) and correlated mean decays per cell using activity concentrations of 6.875 kBq/mL for all drugs (calculated with MIRDcell-Ã) (B). Distances beyond 200 µm represent medium.

Article Snippet: The spheroids comprised the human epidermal growth factor receptor 2–positive human BT474 breast cancer cell line, obtained from the American Type Culture Collection.

Techniques: Liposomes, Activity Assay

Journal: Journal of Nuclear Medicine

Article Title: Preirradiation of Spheroids with 225 Ac-Trastuzumab Improves Penetration of 225 Ac-Liposomes and MIRDcell Predictions of Responses to Drug Cocktails

doi: 10.2967/jnumed.124.269273

Figure Lengend Snippet: MIRDcell calculated mean decays per cell attributed to liposomes in BT474 spheroids based on experimental CFDA-SE liposome penetration data for (100% 225 Ac-liposome case from Howe et al. ) and (50% 225 Ac-liposome, 50% 225 Ac-trastuzumab case from newly retrieved data), and extrapolated/averaged (weighted) liposome penetration data for (70% 225 Ac-liposome, 30% 225 Ac-trastuzumab case) and (30% 225 Ac-liposome, 70% 225 Ac-trastuzumab case).

Article Snippet: The spheroids comprised the human epidermal growth factor receptor 2–positive human BT474 breast cancer cell line, obtained from the American Type Culture Collection.

Techniques: Liposomes

Journal: Journal of Nuclear Medicine

Article Title: Preirradiation of Spheroids with 225 Ac-Trastuzumab Improves Penetration of 225 Ac-Liposomes and MIRDcell Predictions of Responses to Drug Cocktails

doi: 10.2967/jnumed.124.269273

Figure Lengend Snippet: Comparison of BT474 experimental outgrowths taken from Howe et al. to MIRDcell (version 4.14)-predicted SFs as bar plot (A), linear regression plot (B), and Bland–Altman plot (C) (95% limit of agreement, −0.048 to 0.095). Predicted SFs shown were based on 225 Ac-trastuzumab in spheroids with no 225 Ac daughters present and 225 Ac-liposomes in spheroids with 225 Ac daughters present. 225 Ac-trastuzumab and 225 Ac-liposomes in medium both had 225 Ac daughters present. Bars on abscissa of panel A and legend for panels B and C are labeled to denote percentage of activity corresponding to 225 Ac-liposomes and 225 Ac-trastuzumab. For example, “L-100, A-0” is 100% 225 Ac-liposomes and 0% activity on trastuzumab antibodies. Error bars for experimental outgrowths correspond to SD. Final prediction uses penetration data for CFDA-SE liposomes after spheroids are exposed to 6.5 kBq/mL 225 Ac-trastuzumab for 50%/50% case; 70% and 30% 225 Ac-liposome cases use extrapolated/averaged penetration data shown in . For CFDA-SE liposomes, published penetration data that were used for old prediction did not involve pretreatment 225 Ac-trastuzumab. Only final predictions are shown in panels B and C.

Article Snippet: The spheroids comprised the human epidermal growth factor receptor 2–positive human BT474 breast cancer cell line, obtained from the American Type Culture Collection.

Techniques: Comparison, Liposomes, Labeling, Activity Assay

Journal: Journal of Nuclear Medicine

Article Title: Preirradiation of Spheroids with 225 Ac-Trastuzumab Improves Penetration of 225 Ac-Liposomes and MIRDcell Predictions of Responses to Drug Cocktails

doi: 10.2967/jnumed.124.269273

Figure Lengend Snippet: Mean absorbed dose due to 225 Ac-trastuzumab (antibodies) and 225 Ac-liposomes (liposomes) in BT474 spheroids and medium for cells at various radial depths within 200-µm-radius spheroids (top). Below each graph, corresponding MIRDcell 3D slice representation of spheroid is shown for equatorial slices of spheroids. Dark red dots in equatorial slices represent living cells, whereas pink dots represent dead cells.

Article Snippet: The spheroids comprised the human epidermal growth factor receptor 2–positive human BT474 breast cancer cell line, obtained from the American Type Culture Collection.

Techniques: Liposomes

Journal: Journal of Nuclear Medicine

Article Title: Preirradiation of Spheroids with 225 Ac-Trastuzumab Improves Penetration of 225 Ac-Liposomes and MIRDcell Predictions of Responses to Drug Cocktails

doi: 10.2967/jnumed.124.269273

Figure Lengend Snippet: MIRDcell (version 4.16) AI–predicted minimum number of decays necessary to achieve target SF < 0.0001 in BT474 spheroids treated with 225 Ac-trastuzumab (antibodies), 225 Ac-liposomes (liposomes), or combination thereof. 225 Ac daughters were assumed to migrate away from 225 Ac-trastuzumab and to remain near 225 Ac-liposomes within spheroid. MIRDcell AI was limited to maximum molar activity of 10 6 GBq/mol for both drugs and lower limit of 1.17 × 10 5 GBq/mol for antibodies only. No bar appearing for antibodies indicates that 225 Ac-trastuzumab alone could not achieve target SF. When optimized molar activities for combined therapy and liposome-only therapy were checked in Monte Carlo–based MIRDcell simulation, zero cell survivors were achieved for both therapies although liposomes required more total decays to achieve same result. “Liposome with preirradiation” indicates use of penetration profiles after preirradiation with 225 Ac-trastuzumab as in 50%/50% case, and “Liposome without preirradiation” indicates use of liposome penetration profile without preirradiation as in 100% 225 Ac-liposomes case.

Article Snippet: The spheroids comprised the human epidermal growth factor receptor 2–positive human BT474 breast cancer cell line, obtained from the American Type Culture Collection.

Techniques: Liposomes, Activity Assay

Journal: BMC Cancer

Article Title: Protein kinase C-delta inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo

doi: 10.1186/1471-2407-14-90

Figure Lengend Snippet: Effects of PKCδ inhibitors on human cancer stem cells. (A) PCSC and PrCSC cells at 80% confluence were exposed to rottlerin. DMSO served as vehicle control (Vehicle). After 24 and 72 hr of exposure, cell mass was evaluated by MTT assay. Control values were normalized to 100%. p values for comparison between treatments reached significance at 24 hr of exposure (p≤0.01) for both cell types, and remained significant at 72 hr. (B) PCSC and PrCSC cells at 50% confluence were exposed to rottlerin. Cytotoxicity was evaluated by LDH-release assay. Total maximal LDH release was assigned the arbitrary value of 100% (Control). p values for comparison between effects of treatments on LDH release reached significance at 24 hr of exposure (p<0.01) for both cell types, and remained significant at 72 hr. (C) Effects of PKCδ inhibitor on tumor cell clonogenic capacity. PCSC were exposed to vehicle or rottlerin (10 μM) for 6, 18, 24, or 48 hr. Viable cells were enumerated and re-plated in media without inhibitor, and colony numbers were quantitated 15 days later. p values for comparison of treatment effects on clonogenic capacity reached significance (p=0.005) at 18 hr of exposure and remained significant for all subsequent exposure times. The insert is a photograph of stained colonies on plates. (D) Structures of staurosporine, rottlerin, second-generation (KAM1) and third-generation (BJE6-106) derivatives. (E) PCSC and PrCSC cells at 50% confluence were exposed to KAM1 at the indicated concentrations. DMSO served as vehicle control (Vehicle). Cytotoxicity was evaluated by LDH-release assay, as in panel B. p values for comparison between treatment effects on LDH release reached significance at 24 hr of exposure to 2.5 μM KAM1 for PCSC cells and at 10 μM for PrCSC (p≤0.01), and remained significant at 72 hr for all concentrations of KAM1. Error bars represent SEM.

Article Snippet: Breast cancer stem cells (2 × 10 5 ) grown from a metastatic tumor were suspended in human breast cancer stem cell complete growth media (Celprogen, San Pedro, CA) and injected subcutaneous into the right flank of female J:NU mice (The Jackson Laboratory, ME) under anesthesia.

Techniques: MTT Assay, Lactate Dehydrogenase Assay, Staining

Journal: BMC Cancer

Article Title: Protein kinase C-delta inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo

doi: 10.1186/1471-2407-14-90

Figure Lengend Snippet: Effects of PKCδ inhibitors on human tumor cell spheroid formation. (A) Hs578T and MCF7 were plated under adherent or non-adherent conditions. Tumor spheroids and adherent cells were collected at 96 hr, stained for CD24 and CD44, and analyzed by flow cytometry. (B) Hs578T, MCF7, breast cancer stem cells (BCSC) and pancreatic cancer stem cells (PCSC) were plated in tumor spheroid media, in the presence of rottlerin, BJE6-106, or DMSO (Control). Tumor spheroids were enumerated at 96 hr, and normalized to the number of spheroids in the control cultures (assigned an arbitrary value of 100%). p values for comparison between vehicle and rottlerin or BJE6-106 effects were significant (p≤0.001). Photographs are of representative areas of the culture plates. (C) MCF7 cells were exposed BJE6-106 or to rottlerin at the indicated concentrations. The corresponding equivalent volume of solvent (DMSO) was used as a vehicle control (Vehicle). After 24, 48 and 72 hr of exposure, cell mass was evaluated by MTT assay. Control values were normalized to 100%. p values for comparison between vehicle and rottlerin effects on cell number at 24 hr reached significance at 5 μM, and for BJE6-106 at 0.5 μM (p ≤ 0.02), and were significant for all concentrations tested at 48 and 72 hr time points. (D) Hs578T cells were exposed to vehicle or BJE6-106 (1 μM) for 6, 12, 24, 48 or 96 hr. Viable cells were enumerated and re-plated in media without BJE6-206, and spheroid numbers were quantitated 96 hr later. p values for comparison between vehicle and BJE6-106 effects on spheroid number were significant after 6 hr of exposure (p≤0.02), and remained significant at all time points thereafter. Error bars represent SEM.

Article Snippet: Breast cancer stem cells (2 × 10 5 ) grown from a metastatic tumor were suspended in human breast cancer stem cell complete growth media (Celprogen, San Pedro, CA) and injected subcutaneous into the right flank of female J:NU mice (The Jackson Laboratory, ME) under anesthesia.

Techniques: Staining, Flow Cytometry, MTT Assay

Journal: BMC Cancer

Article Title: Protein kinase C-delta inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo

doi: 10.1186/1471-2407-14-90

Figure Lengend Snippet: Effects of PKCδ inhibitors on growth and spheroid formation in non-transformed and transformed human breast epithelial cells. MCF 10A cells and cells from the derived tumorigenic line MCF 10C (also called M3), were grown to 80% confluence in 96-well plates and then exposed to rottlerin at concentrations ranging from 1 to 20 μM (A) or to BJE6-106 at concentrations ranging from 0.1 to 20 μM (B) . The corresponding equivalent volume of solvent (DMSO) was used as a vehicle control (Vehicle). After 24, 48 and 72 hr of exposure, cell mass was evaluated by MTT assay. Control (vehicle) values were normalized to 100%. Error bars represent SEM. p values for comparison between vehicle and PKCδ inhibitors on MCF 10A cell number only reached significance (p < 0.05) at 48 hr at 20 μM for rottlerin, and at 1 μM for BJE6-106. In contrast, significant effects of the inhibitors on the MCF 10C cells were observed as early as 24 hr for rottlerin (at 5 μM) and for BJE6-106 (at 0.1 μM). (C) MCF 10A and MCF 10C cells were plated at 10,000 cells per well in tumor spheroid media, and spheroid formation was assessed at days 10 and 21. Representative photographs are shown. (D) MCF 10C cells were plated at 10,000 cells per well in tumor spheroid media, in the presence of rottlerin (5 μM), or BJE6-106 (1 μM or 5 μM), or DMSO vehicle (Control). Tumor spheroids were enumerated at 10 days. Representative photographs are shown. (E) Spheroid numbers were normalized to the number of spheroids in the control cultures (assigned an arbitrary value of 100%) and plotted. Error bars represent SEM. p values for comparison between vehicle and rottlerin or BJE6-106 effects on spheroid number were significant (p < 0.001).

Article Snippet: Breast cancer stem cells (2 × 10 5 ) grown from a metastatic tumor were suspended in human breast cancer stem cell complete growth media (Celprogen, San Pedro, CA) and injected subcutaneous into the right flank of female J:NU mice (The Jackson Laboratory, ME) under anesthesia.

Techniques: Transformation Assay, Derivative Assay, MTT Assay

Journal: BMC Cancer

Article Title: Protein kinase C-delta inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo

doi: 10.1186/1471-2407-14-90

Figure Lengend Snippet: Effects of PKCδ inhibitor on tumor growth and survival in a xenograft human breast cancer stem cell model. Breast cancer stem cell xenografts were established and animals were treated with vehicle or rottlerin for 15 days, as described in Methods. (A) Tumor volumes plotted over time, until tumors in all the control animals reached the maximum volume allowed by the protocol (approximately 15 days). (B) Kaplan-Meier plot of survival of vehicle control or rottlerin (PKCδi)-treated animals, with monitoring continuing after cessation of treatment at day 15.

Article Snippet: Breast cancer stem cells (2 × 10 5 ) grown from a metastatic tumor were suspended in human breast cancer stem cell complete growth media (Celprogen, San Pedro, CA) and injected subcutaneous into the right flank of female J:NU mice (The Jackson Laboratory, ME) under anesthesia.

Techniques:

Journal: BMC Cancer

Article Title: Protein kinase C-delta inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo

doi: 10.1186/1471-2407-14-90

Figure Lengend Snippet: Immunoblot analysis of Ras-signaling pathways in tumor cells, cell lines, or spheres. Hs578T, MCF7, MDA231 and breast cancer stem cells (BCSC) were plated in tumor spheroid media under adherent or non-adherent conditions. Tumor spheroids and adherent cells were collected at 96 hr, and lysed. MCF 10A (M1) and MCF 10C (M3) lysates were also prepared. The lysates were separated by electrophoresis and immunoblotted with antibodies against ERK, pEKR, AKT, pAKT, JNK, pJNK. Immunoblotting of α-tubulin serves as a loading control. For quantitation, the digital intensity of the bands was first normalized to α-tubulin in each lane, and then expressed relative to the signal for the MCF 10A (M1) cell line. Values are shown under each band.

Article Snippet: Breast cancer stem cells (2 × 10 5 ) grown from a metastatic tumor were suspended in human breast cancer stem cell complete growth media (Celprogen, San Pedro, CA) and injected subcutaneous into the right flank of female J:NU mice (The Jackson Laboratory, ME) under anesthesia.

Techniques: Western Blot, Electrophoresis, Quantitation Assay

Journal: PLoS ONE

Article Title: Induction of AMPK activation by N , N’- diarylurea FND-4b decreases growth and increases apoptosis in triple negative and estrogen-receptor positive breast cancers

doi: 10.1371/journal.pone.0209392

Figure Lengend Snippet: Equal numbers of MCF-7, T-47D, MDA-MB-231, HCC-1806 or breast cancer stem cells were treated with fresh medium that contained different FND-4b concentrations (0, 1, 2.5, 5, 10, and 20 μM) for 24 h. Western blot analysis was then performed for phosphorylated and total forms of AMPKα, ACC, and S6 as well as cyclin D1 and cleaved PARP. Beta-actin was used as the loading control. Treatment of the HCC-1806 cell line was repeated to confirm the spike in AMPKα activation at the 5 μM dosage with subsequent decreases at higher concentrations.

Article Snippet: Human breast cancer stem cell complete growth medium was from Celprogen (Torrance, CA).

Techniques: Western Blot, Activation Assay

Journal: PLoS ONE

Article Title: Induction of AMPK activation by N , N’- diarylurea FND-4b decreases growth and increases apoptosis in triple negative and estrogen-receptor positive breast cancers

doi: 10.1371/journal.pone.0209392

Figure Lengend Snippet: (A) Equal numbers of MCF-7, T-47D, MDA-MB-231, HCC-1806, or breast cancer stem cells were grown in medium containing 0 or 5 μM FND-4b for 72 h followed by cell counting. (B) The same procedure described in (A) was followed with the exception of growing cells in medium containing 0 or 1 mM AICAR instead. (C) MCF-7, T-47D, MDA-MB-231, and HCC-1806 cells were grown in medium containing different concentrations of FND-4b (0, 2.5, 5, 10, and 20 μM) for 72 h before SRB growth assays were performed. Data are presented as mean ± SD from experiments performed in triplicate (cell counting) or sextuplicate (SRB assays) and are representative of three independent experiments. * indicates p-value < 0.01.

Article Snippet: Human breast cancer stem cell complete growth medium was from Celprogen (Torrance, CA).

Techniques: Cell Counting