Fig 1: In vitro, MDA-MB-231.CCR5−/− cells are less metabolically active than MDA-MB-231.CCR5+/+ cells. a Cells were either left untreated (medium alone), treated with 10 nM CCL5, or pre-treated with 2 mM 2-DG for 1 h prior to CCL5 treatment, or maintained in medium containing 5 mM glutamine. For all, medium was changed every other day and the treatment(s) reapplied. Cell proliferation was quantified using an MTT assay as described in Methods. The proliferation index is normalized against untreated conditions (ie medium alone). Values are means ± SEM of triplicate assays and each data point combines the data from 3 independent experiments. Statistical analysis was performed comparing untreated cells with CCL5-treated cells and inhibitor-treated cells with CCL5 + inhibitor treated cells, or comparing CCL5-treated with CCL5 + inhibitor-treated cells. b CCL5 levels were measured from culture supernatants after 16 h incubation. c Glucose uptake, d GLUT-1 expression, e intracellular ATP and f intracellular lactate were measured as described in Methods in cells treated with 10 nM CCL5 or 2 μM oligomycin for 3 h. Data are expressed as percent-change relative to untreated MDA-MB-231.CCR5+/+ cells. Values are means ± SEM of triplicate assays and each data point combines the data from 3 independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001
Fig 2: MMTV-PyMT.CCR5−/− tumor cells harvested from NSG mice exhibit lower glucose uptake, reduced GLUT-1 expression and lower levels of cellular metabolites than MMTV-PyMT.CCR5+/+ tumor cells. MMTV-PyMT.CCR5+/+ and MMTV-PyMT.CCR5−/− tumors were harvested from recipient NSG mice when they were either just palpable (~20mm3), i.e. tumor onset, or ~700mm3, endpoint. Following 16 h in culture, a glucose uptake, b GLUT-1 expression, c intracellular lactate and d CCL5 production, were measured. Values are the means +/− SEM of technical triplicates. *p < 0.05, ** p < 0.01, *** p < 0.001
Fig 3: MMTV-PyMT.CCR5−/− mice have delayed tumor onset and increased tumor free survival compared to MMTV-PyMT.CCR5+/+ mice. a, b MMTV-PyMT.CCR5+/+ and MMTV-PyMT.CCR5−/− mice were monitored daily for palpable tumors, using calipers. Tumor onset is defined as detection of a palpable tumor, volume ~20mm3. Tumor endpoint is defined as a palpable tumor with volume ~700mm3. Mice were euthanized when tumors reached this size. Each data point represents a single mouse. Means and SEM are identified as horizontal lines. c Tumor-free survival rate is the percentage of the mice in the colony that are tumor-free at each specified age. * * p < 0.01
Fig 4: MMTV-PyMT.CCR5−/− tumor cells transplanted into NSG mice exhibit delayed tumor growth compared with MMTV-PyMT.CCR5+/+ tumor cells. Tumors (100-700 mm3) from MMTV-PyMT.CCR5+/+ and MMTV-PyMT.CCR5−/− mice were harvested and injected into the lower mammary fat pads of NSG recipients (n = 12), as described in Methods. Tumor growth was monitored externally using calipers. a Time to palpable tumor onset (~20mm3) and b tumor endpoint (~700mm3). Each data point represents a single mouse. c Tumor volume was measured over time to determine the rate of tumor growth. Means and SEM are identified as horizontal lines. d Onset tumors were harvested, fixed, thin sections prepared and stained with H & E. Arrows show indicated features. e The average number of mitotic figures in each of 5 fields/thin section at 20× magnification are recorded. * p < 0.05 and *** p < 0.001
Fig 5: MDA-MB-231.CCR5−/− cells transplanted in NSG mice exhibit delayed tumor growth compared with MDA-MB-231.CCR5+/+ cells. MDA-MB-231.CCR5−/− and MDA-MB-231.CCR5+/+ cells were injected into mammary fat pads of NSG mice (n = 12), as described in Methods. Tumor growth was monitored externally using calipers. a Time to palpable tumor onset (~20mm3). Each data point represents a single mouse. b Tumor volume was measured over time to determine tumor growth. Means and SEM are identified as horizontal lines. c Onset tumors were frozen, thin sections prepared and stained with H & E. Arrows show indicated features. d The average number of mitotic figures in each of 5 fields/thin section at 20× magnification are recorded.* p < 0.05
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