Sequential Colocalization of ERa, PR, and AR Hormone Receptors Using Confocal Microscopy Enables New Insights into Normal Breast and Prostate Tissue and Cancers

Abstract

Multiplex immunohistochemistry (mIHC) use markers staining different cell populations applying widefield optical microscopy. Resolution is low not resolving subcellular co-localization. We sought to colocalize markers at subcellular level with antibodies validated for clinical diagnosis, including the single secondary antibody (combination of anti-rabbit/mouse-antibodies) used for diagnostic IHC with any primary antibody, and confocal microscopy. We explore colocalization in the nucleus (ColNu) of nuclear hormone receptors (ERa, PR, and AR) along with the baseline marker p63 in paired samples of breast and prostate tissues. We established ColNu mIHCF as a reliable technique easily implemented in a hospital setting. In ERa+ breast cancer, we identified different colocalization patterns (nuclear or cytoplasmatic) with PR and AR on the luminal epithelium. A triple-negative breast-cancer case expressed membrane-only ERa. A PR-only case was double positive PR/p63. In normal prostate, we identified an ERa+/p63+/AR-negative distinct population. All prostate cancer cases characteristically expressed ERa on the apical membrane of the AR+ epithelium. We confirmed this using ERa IHC and needle-core biopsies. ColNu mIHCF is feasible and already revealed a new marker for prostate cancer and identified sub-patterns in breast cancer. It could be useful for pathology as well as for functional studies in normal prostate and breast tissues.

Keywords: TSA; confocal microscopy; membrane estrogen receptor alpha.

Figure 1

ColNu mIHCF for nuclear hormone receptors with negative controls for each cycle. (A) Confocal maximal projections after sequential staining for ERa, PR, and AR in normal breast tissue performed on one slide while omitting one or all primary antibodies (Cneg) in parallel slides. (B) Overlay of the channels with DAPI (top) or phase contrast (DIC, bottom). * white asterisk indicates air bubble in the mounting medium. Scale bar shown in DAPI channel. (C) Quantification using Fiji or a designed pipeline with Cell Profiler. No differences were found in single channels in normal breast or prostate. Cell profiler was more sensitive for double or triple colocalization. The order of antibodies in the consecutive cycles is indicated above each photograph. Pseudocolor was kept constant: green for the 1st, red for the 2nd, and blue for the 3rd cycle. DAPI was included in the mounting medium and reveals the nuclei. DIC is a plane obtained with transillumination for differential interfering contrast-Nomarski and reveals tissue structure.

Figure 2

ColNu mIHCF in normal breast tissue from paired cancer cases. (A) NB5 reveals some epithelial cells co-expressing ERa + PR; others co-express ERa + AR, while many are single positive for ERa, PR, or AR. Stroma was positive for PR. (B) NB1 shows many epithelial cells single positive for ERa, with some colocalizing ERa + AR. PR+ cells accumulated in the stroma, suggesting an early alteration. The tissue maintained the basal p63 layer (C) showing some positivity for PR. (D,E) NB4 also presents many cells co-expressing ERa + PR (D) and others co-expressing ERa + AR (E), while the basal layer is negative for hormone receptors. TTF1 is negative in the epithelium and inconsistently positive in the stroma. (F) NB3 shows hyperplasia of the ERa epithelium with some population co-expressing ERa + AR. The basal p63 layer, negative for hormone receptors, is maintained. The order of antibodies in the consecutive cycles is indicated above each photograph. Pseudocolor was kept constant: green for the 1st, red for the 2nd, and blue for the 3rd cycle. DAPI was included in the mounting medium and reveals the nuclei. DIC is a plane obtained with transillumination for differential interfering contrast-Nomarski and reveals tissue structure.

Figure 3

ColNu mIHCF in breast cancer from different hormone receptor and HER2 status. (A,B) BC1 presents ductal carcinoma in situ (DCIS) area maintaining the basal p63 layer. Epithelium co-expresses nuclear ERa with strong whole-cell (cytoplasmic + nucleus) PR and AR localization. In A (top-left), the blended white color indicates overlay in quantity and location of all three receptors. (C,D) BC3 is another DCIS area with an intact p63 basal layer. All three receptors, ERa + PR + AR, are co-expressed exclusively in the nucleus, as shown by the blended yellow color in ER + PR (B) and in ER + AR (D). TTF1 was negative, as were all the other breast cancer cases. (E,F) BC2 epithelium (invasive ductal carcinoma) expresses PR exclusively, being negative for both ERa and AR. The PR+ population presents a p63+ sub-population. (G,H) BC4, a triple negative invasive ductal carcinoma, shows negativity for all three nuclear receptors. The cancer epithelium expresses p63. Shown are two independent fields for each multiple combination of antibodies. The order of antibodies in the consecutive cycles is indicated above each photograph. Pseudocolor was kept constant: green for the 1st, red for the 2nd, and blue for the 3rd cycle. DAPI was included in the mounting medium and reveals the nuclei. DIC is a plane obtained with transillumination for differential interfering contrast-Nomarski and reveals tissue structure.

Figure 4

A breast cancer case diagnosed as triple negative presents plasma membrane ERa expression in ColNu mIHCF. BC5 is a case of ERa/PR-negative invasive ductal carcinoma with low HER2 positivity, and negative FISH. (A,B) In DCIS areas, ColNu mIHCF reveals epithelium with ERa located on the plasma membrane, strong nuclear AR with some PR colocalization, and PR also located on membrane regions not in contact with other epithelial cells. A basal layer of cells in contact with the stroma was strongly PR+. (C) Tumor cells also shows express p63. At the stroma, the most abundant population is PR+ at a distance from the cancer (A,B) and p63 in the vicinity (C). The order of antibodies in the consecutive cycles is indicated above each photograph. Pseudocolor was kept constant: green for the 1st, red for the 2nd, and blue for the 3rd cycle. DAPI was included in the mounting medium and reveals the nuclei. DIC is a plane obtained with transillumination for differential interfering contrast-Nomarski and reveals tissue structure.

Figure 4

A breast cancer case diagnosed as triple negative presents plasma membrane ERa expression in ColNu mIHCF. BC5 is a case of ERa/PR-negative invasive ductal carcinoma with low HER2 positivity, and negative FISH. (A,B) In DCIS areas, ColNu mIHCF reveals epithelium with ERa located on the plasma membrane, strong nuclear AR with some PR colocalization, and PR also located on membrane regions not in contact with other epithelial cells. A basal layer of cells in contact with the stroma was strongly PR+. (C) Tumor cells also shows express p63. At the stroma, the most abundant population is PR+ at a distance from the cancer (A,B) and p63 in the vicinity (C). The order of antibodies in the consecutive cycles is indicated above each photograph. Pseudocolor was kept constant: green for the 1st, red for the 2nd, and blue for the 3rd cycle. DAPI was included in the mounting medium and reveals the nuclei. DIC is a plane obtained with transillumination for differential interfering contrast-Nomarski and reveals tissue structure.

Figure 5

ColNu mIHCF in normal (non-malignant) prostatic tissue from paired cancer cases identifies a basal cell co-expressing p63 and ERa. (A) NP2 illustrates the prostate epithelium expressing AR with a few isolated ERa+/AR− epithelial cells. PR is expressed at the stroma together with some ERa positive cells. (B) ERa epithelial cells are basal cells co-expressing p63 as seen by the blended yellow color. Few of these cells express greater amounts of ERa than p63, resulting in bright green. The basal layer is p63+, but AR-. The same pattern is repeated in cases NP3 (C,D) and NP4 (E,F). (G) NP5 illustrates a different staining pattern in which the AR+ epithelial cells co-express PR. In addition to the few basal ERa+ cells, a layer of ERa+ elongated cells is also observed. This sample was considered prostate hyperplasia. (H) The two types of ERa+ cells are shown at lower magnification. Isolated basal epithelial cells co-expressing p63 and ERa and another layer of elongated ERa in the proximity of the basal layer. (I) At high magnification, the prostate epithelium expressing PR, the p63 layer with discrete ERa+ cells, and the second layer of ERa elongated cells p63− can be seen. The order of antibodies in the consecutive cycles is indicated above each photograph. Pseudocolor was kept constant: green for the 1st, red for the 2nd, and blue for the 3rd cycle. DAPI was included in the mounting medium and reveals the nuclei).

Figure 5

ColNu mIHCF in normal (non-malignant) prostatic tissue from paired cancer cases identifies a basal cell co-expressing p63 and ERa. (A) NP2 illustrates the prostate epithelium expressing AR with a few isolated ERa+/AR− epithelial cells. PR is expressed at the stroma together with some ERa positive cells. (B) ERa epithelial cells are basal cells co-expressing p63 as seen by the blended yellow color. Few of these cells express greater amounts of ERa than p63, resulting in bright green. The basal layer is p63+, but AR-. The same pattern is repeated in cases NP3 (C,D) and NP4 (E,F). (G) NP5 illustrates a different staining pattern in which the AR+ epithelial cells co-express PR. In addition to the few basal ERa+ cells, a layer of ERa+ elongated cells is also observed. This sample was considered prostate hyperplasia. (H) The two types of ERa+ cells are shown at lower magnification. Isolated basal epithelial cells co-expressing p63 and ERa and another layer of elongated ERa in the proximity of the basal layer. (I) At high magnification, the prostate epithelium expressing PR, the p63 layer with discrete ERa+ cells, and the second layer of ERa elongated cells p63− can be seen. The order of antibodies in the consecutive cycles is indicated above each photograph. Pseudocolor was kept constant: green for the 1st, red for the 2nd, and blue for the 3rd cycle. DAPI was included in the mounting medium and reveals the nuclei).

Figure 6

ColNu mIHCF in prostate cancer reveals ERa at the apical membrane of cancer epithelium. (A) ColNu mIHCF for all three nuclear receptors in PC1 showing each separate channel (center) and the overlays among them (left) or with DAPI (right). Prostate cancer epithelium expresses AR (blue) in the nucleus, but ERa (green) on the apical membrane. PR is not expressed. (B–D) Lower magnifications reveal the same pattern of nuclear AR and apical membrane ERa co-expression in PC1 (B), PC2 (C), and PC4 (D). (E) Similar staining with different order and TSA for AR, again exhibits nuclear AR (red) and apical membrane ERa (green) co-expression in the cancer epithelium of case PC5. TTF1 staining was negative. (F,G) A phase contrast plane (DIC) is shown for paired samples of case NP1/PC1 alone or overlaid with other channels. A single basal ERa cell is shown underneath the normal prostate epithelium expressing nuclear AR in NP1 (F). The whole prostate cancer epithelium co-expressing nuclear AR and apical membrane ERa is shown in PC1 (G). (H) At lower magnifications (case PC3), the combination of p63 and ERa demarcate prostate cancer from normal prostate tissue. On the left, prostate cancer with ERa located on the apical membrane. On the right, normal prostate epithelium with the p63 basal layer displaying some p63/ERa double positive cells. PR is expressed at the stroma. (I) The same result was obtained for PC3 and PC4. While AR is located in the nucleus of both cancer and normal prostate epithelial cells, apical membrane ERa staining is specific for cancer, while p63 is conserved at the intact basal layer of normal prostate tissue. The order of antibodies in the consecutive cycles is indicated above each photograph. Pseudocolor was kept constant: green for the 1st, red for the 2nd, and blue for the 3rd cycle. DAPI was included in the mounting medium and reveals the nuclei. DIC is a plane obtained with transillumination for differential interfering contrast-Nomarski and reveals tissue structure).

Figure 7

Immunohistochemistry with ColNu mIHCF conditions confirms ERa as a marker of prostate cancer. (A) Representative microphotographs of prostate cancers from TMA-I and II performed with overnight incubation of the primary antibody (ColNu mIHCF conditions). Left: Cancer epithelium is positive for ERa at the apical membrane, while normal prostate epithelium is not. Right: Nuclei of cancer and normal epithelial cells are positive for AR. (B) Left: Normal prostate epithelium is negative for ERa. At the basal layer, isolated cells show positivity for ERa. Right: Prostate epithelial cells in normal tissue express nuclear AR. (C) Sections from prostate needle-core biopsies similarly stained for ERa showing the stained prostate cancer while the normal tissue (asterisk) is negative or presents isolated nuclear ERa positive cells at the basal layer. Right: a case with no malignancy exhibits a proliferation of ERa cells within the stroma. (*, asterisks indicate normal prostate epithelium).