Molecular heterogeneity in adjacent cells in triple-negative breast cancer

Figures & data

Table 1 Hyperspectral immunologic panels

Dye	Target	Panel					Pos control	Neg control
		1	2	3	4	5
1. DAPI	DNA	x	x	x	x	x
2. AF488	Cytokeratin	x	x	x	x	x	SKBr3	PBMC
3. AF532	ALDH-1	x	x			x	HepG2	PBMC
4. AF555	NOTCH1			x	x		SKBr3	PBMC
5. AF546	CD24	x	x			x	HepG2	PBMC
6. AF568	CD44	x	x			x	MCF-7	PBMC
7. AF594	uPAR	x		x			SKBr3/MCF-7	Raji
	CD133		x		x	x	MCF-7	PBMC
8. AF633	CD45	x	x				PBMC	A549
	Notch1					x	SKBr3	PBMC
9. AF647	Ki67	x	x				Colo205	BT474
10. AF660	Her-2	x	x				MCF-7	PBMC
11. AF700	Twist

Notes: AF594 and AF633 were conjugates of two different antibodies, which create different panels. Positive and negative control columns list the cell lines used in determining the amount of the dye–target conjugate to measure the overexpression. The AF700/twist conjugate was not yet applied.

Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole, dihydrochloride; ALDH-1, acetaldehyde dehydrogenases 1; uPAR, urokinase plasminogen activator surface receptor; AF, AlexaFluor; PBMC, peripheral blood mononuclear cells; Notch1, notch homolog 1; CD, cluster of differentiation; Her-2, human epidermal growth factor receptor 2; Pos, positive; Neg, negative.

Figure 1 Comparison of expression heterogeneity using the distribution variance with patient tumor size for the markers: ALDH1, uPAR, and Her-2.

Notes: Marker variance, as a measure for heterogeneity, compared to tumor size. Least squares fit line to the data points also plotted. Heterogeneity in marker overexpression increases with tumor size in BC patients while heterogeneity in TN patients decreases with tumor size.
Abbreviations: ALDH1, acetaldehyde dehydrogenases 1; uPAR, urokinase plasminogen activator surface receptor; Her-2, human epidermal growth factor receptor 2; BC, breast cancer; TN, triple-negative; LSfit, least squares fit; Cts/s/pxl, counts/second/pixel.

Table 2 Fraction of overexpressed cells for each marker

Marker	Overexpressed BC (%)					Overexpressed TN (%)
	Patient					Patient
	1	2	3	4	All	1	2	3	4	5	6	All
ALDH1
OE	16	13	37	49	25	51	60	39	73	36	61	51
HiOE	3	3	0	25	4	15	34	0	27	13	19	16
CD24
OE	16	12	23	35	19	29	42	1	13	13	33	26
HiOE	0	0	0	4	1	13	33	0	0	0	9	13
CD44
OE	24	3	51	27	27	42	55	17	87	24	60	42
HiOE	0	1	0	2	1	18	34	0	7	4	11	16
Notch1
OE	NA	NA	NA	NA	NA	NA	13	NA	NA	NA	NA	13
HiOE	NA	NA	NA	NA	NA	NA	7	NA	NA	NA	NA	7
uPAR
OE	86	62	90	NA	75	81	89	100	NA	NA	89	85
HiOE	10	0	0	NA	2	15	9	0	NA	NA	11	11
CD133
OE	NA	NA	0	42	14	34	28	3	7	20	14	24
HiOE	NA	NA	0	38	13	13	2	0	0	0	0	6
Ki67
OE	NA	NA	NA	NA	NA	28	2	0	0	18	24	19
HiOE	NA	NA	NA	NA	NA	3	0	0	0	0	1	2
Her-2
OE	2	16	1	49	11	19	6	0	0	0	0	11
HiOE	0	0	0	2	0	1	0	0	0	0	0	1

Notes: Percentage of measured cells with overexpressed (2+) and highly overexpressed (3+) markers. Tabulated by marker for each patient, including the average for each patient group. Currently, only one patient has been assayed with a panel including Notch1. None of the BC patients had been assayed with a Ki67 panel.

Abbreviations: BC, breast cancer; TN, triple-negative; OE, overexpressed (2+); HiOE, highly overexpressed (3+); ALDH1, acetaldehyde dehydrogenases 1; CD, cluster of differentiation; uPAR, urokinase plasminogen activator surface receptor; Her-2, human epidermal growth factor receptor 2; Notch1, notch homolog 1; NA, not applicable.

Figure 2 Cell marker expression of ALDH1, uPAR, CD133, and Her-2.

Notes: All cells plotted for: (A) ALDH1, (B) uPAR, (C) CD133, and (D) Her-2. Measurements are plotted in the order the cells were scanned. Solid horizontal lines indicate the emission threshold for overexpressed. Dashed-dotted horizontal lines indicate twice as bright as the threshold expression. *The “n” indicates that the examined cells demonstrate no stem cell markers.
Abbreviations: ALDH1, acetaldehyde dehydrogenases 1; uPAR, urokinase plasminogen activator surface receptor; CD133, cluster of differentiation 133; Her-2, human epidermal growth factor receptor 2; CSC, cancer stem cells; Cts/s/pxl, counts/second/pixel; BC, breast cancer; TN, triple-negative.

Figure 3 CD44 versus CD24 expression for all cells for both hormonal receptor positive and TNBC patients.

Notes: Solid horizontal and vertical lines indicate the emission threshold for overexpressed. Dashed-dotted horizontal and vertical lines indicate twice as bright as threshold overexpressed. Diagonal solid line indicates the cells which have higher expression in one antibody over the other (ie, all CD44+/CD24− are above the line). The TN cell, plotted in red, shows a larger heterogeneity than the BC cells, plotted in blue. *The “n” indicates that the examined cells demonstrate no stem cell markers.
Abbreviations: BC, breast cancer; TN, triple-negative; CSC, cancer stem cells; CD, cluster of differentiation; Cts/s/pxl, counts/second/pixel.

Figure 4 Comparison of cell expression of ALDH1 versus uPAR.

Notes: ALDH1 expression compared to uPAR expression for each cell. Plot symbols group data by BC and TN patients and subgroup by CSC and non-CSC. Trend lines of the data subgroups indicate higher heterogeneity in BC and TN patient cells, which have the stem cell CD44+/CD24− character. *The “n” indicates that the examined cells demonstrate no stem cell markers.
Abbreviations: ALDH1, acetaldehyde dehydrogenases 1; uPAR, urokinase plasminogen activator surface receptor; BC, breast cancer; TN, triple-negative; CSC, cancer stem cells; CD, cluster of differentiation; Cts/s/pxl, counts/second/pixel.