**3. Application area**

The identification of estrogen receptor (ER) and progesterone receptor (PR) in the current testing of breast cancer has advanced the field. Other hormones/growth factors are also involved; for example, PRL and its receptor isoforms. If their roles are more clearly identified with the use of specific antibodies then there may be a practical need for them in the diagnosis of this disease. Examining normal breast development could give clues to the relevance of PRLR isoforms in cancer. Not only do these apply to the breast, but may also be of value to studying ovarian and prostate cancers where PRL may play a role/function. PRLR isoform specific antibodies could be powerful tools in this quest.

#### **4. Methods**

#### **4.1. Preparation of the PRLR isoform specific monoclonal antibodies**

Synthetic peptides were designed based on the regions of unique intracellular sequences of the PRLR splice variants and synthesized (AnaSpec, Inc., San Jose, CA) by the solid-phase method. Peptides (4 mg) were conjugated to bovine serum albumin (10 mg) with 1mM DSS overnight at room temperature and sent to Epitomics (Burlingame, CA) for immunization of rabbits. Initially, a total of 76 clones were received (54 clones for SF1a, 22 clones for SF1b) and screened for reactivity to their respective antigens by Western blot analysis. Briefly, lysates of CHO-K1 cells expressing either SF1a or SF1b were separated by SDS-PAGE, and transferred to PVDF. Membranes were blocked for 1 hr at room temperature in 5% non-fat dairy milk diluted in TBST+0.1% sodium azide. Subsequently, the clonal supernatants were diluted 1:1 in 10% non-fat dairy milk (diluted in TBST+0.1% sodium azide) and incubated for 3 hr at room temperature. Membranes were washed and probed with goat anti-rabbit Alexa680 (1:4000) prior to visualization using a LiCOR Odyssey reader. Supernatants from positively reacting clones were re-screened prior to selection for subcloning. In total, four SF1a and six SF1b rabbit monoclonal antibodies were put into production. The rabbit sera were initially purified by MEP Hypercel (PAL) chromatography, followed by size-exclusion chromatography to further purify the antibodies. Final pools of antibody were produced and protein concentration determined by the Pierce 660nm assay.

For LF, 6 mg peptide was conjugated to 3 mg keyhole limpet hemocyanin overnight at room temperature using glutaraldehyde. Peptide conjugates were sent to Green Mountain Antibodies (Burlington, VT) for generation of mouse monoclonal antibodies. Initially, 120 clones were screened by Western blot as described above using CHO-K1 cells expressing LF with the exception that the antibodies were diluted 1:100 in 5% non-fat dairy milk (diluted in TBST+0.1% sodium azide) and incubated overnight at 4o C. Secondary antibody and imaging were essentially as described above. Supernatants of positively reacting clones were re-screened by Western blot and examined by fluorescence microscopy (to confirm appropriate localization) prior to selection for production. In total, 10 positive clones were selected for this process, 8 of which produced IgG. Antisera was purified as described for SF1a and SF1b antibodies. Final pools of antibody were produced and protein concentration determined by the Pierce 660nm assay.

#### **4.2. Cell culture and transfection**

196 Prolactin

revealed that the three isoforms, LF, SF1a, and SF1b, are differentially expressed in ductal and lobular carcinomas [5]. These two most common histological types of breast cancer originate from the terminal ductal lobular unit and may be difficult to classify. However, distinct differences were observed in PRLR expression in normal, benign, and malignant breast tissue which may have prognostic significance [10, 11]. The development and characterization of PRLR isoform specific monoclonal antibodies will provide a near limitless supply of reagent to continue to examine how and where these isoforms interact

The identification of estrogen receptor (ER) and progesterone receptor (PR) in the current testing of breast cancer has advanced the field. Other hormones/growth factors are also involved; for example, PRL and its receptor isoforms. If their roles are more clearly identified with the use of specific antibodies then there may be a practical need for them in the diagnosis of this disease. Examining normal breast development could give clues to the relevance of PRLR isoforms in cancer. Not only do these apply to the breast, but may also be of value to studying ovarian and prostate cancers where PRL may play a role/function.

Synthetic peptides were designed based on the regions of unique intracellular sequences of the PRLR splice variants and synthesized (AnaSpec, Inc., San Jose, CA) by the solid-phase method. Peptides (4 mg) were conjugated to bovine serum albumin (10 mg) with 1mM DSS overnight at room temperature and sent to Epitomics (Burlingame, CA) for immunization of rabbits. Initially, a total of 76 clones were received (54 clones for SF1a, 22 clones for SF1b) and screened for reactivity to their respective antigens by Western blot analysis. Briefly, lysates of CHO-K1 cells expressing either SF1a or SF1b were separated by SDS-PAGE, and transferred to PVDF. Membranes were blocked for 1 hr at room temperature in 5% non-fat dairy milk diluted in TBST+0.1% sodium azide. Subsequently, the clonal supernatants were diluted 1:1 in 10% non-fat dairy milk (diluted in TBST+0.1% sodium azide) and incubated for 3 hr at room temperature. Membranes were washed and probed with goat anti-rabbit Alexa680 (1:4000) prior to visualization using a LiCOR Odyssey reader. Supernatants from positively reacting clones were re-screened prior to selection for subcloning. In total, four SF1a and six SF1b rabbit monoclonal antibodies were put into production. The rabbit sera were initially purified by MEP Hypercel (PAL) chromatography, followed by size-exclusion chromatography to further purify the antibodies. Final pools of antibody were produced

For LF, 6 mg peptide was conjugated to 3 mg keyhole limpet hemocyanin overnight at room temperature using glutaraldehyde. Peptide conjugates were sent to Green Mountain

both in normal breast development and in breast cancer.

PRLR isoform specific antibodies could be powerful tools in this quest.

and protein concentration determined by the Pierce 660nm assay.

**4.1. Preparation of the PRLR isoform specific monoclonal antibodies** 

**3. Application area** 

**4. Methods** 

Chinese hamster ovary cells (CHO-K1, ATCC, Manassas, VA) were maintained in -MEM (Invitrogen, Gaithersburg, MD) supplemented with 5% fetal bovine serum (FBS, Invitrogen) and penicillin/streptomycin (100 U/ml and 100 g/ml respectively, Invitrogen). Transfections were performed using FuGENE 6 (Roche Applied Science, Indianapolis, IN) at a ratio of 1 g DNA to 3 l FuGENE. The PRLR isoform specific cDNA constructs were previously described [1]. Cells were transfected for 48 hr, then allowed to grow for an additional 48 hr.

T47D, ZR75-1, MDA-MB-231, MDA-MB-468, MCF7, and SK-BR-3 breast cancer cell lines were obtained from ATCC. T47D, ZR75-1, MDA-MB231, and MDA-MB468 cells were maintained in RPMI1640 (Invitrogen). MCF7 cells were grown in DMEM and SK-BR-3 cells were maintained in McCoys 5a media. All media were supplemented with 5% heat inactivated FBS, 10 g/ml bovine insulin (Sigma, St. Louis, MO), and penicillin-streptomycin (100 U/ml and 100 g/ml respectively, Invitrogen). Where indicated, breast cancer cell lines were plated on 8-well glass chamber slides (Nunc, Rochester, NY) in normal growth media, and allowed to attach overnight. The media were replaced and the cells were treated for the indicated times with 500 ng/ml recombinant human PRL in media where the FBS was replaced with 1% charcoal stripped serum, then fixed in 10% normal buffered formalin prior to Duolink assay.

All cells were maintained at 37 °C in a humidified atmosphere with 5% CO2. Cells were passaged using trypsinization (0.05% trypsin-EDTA, Invitrogen) and counted on a hemocytometer using trypan blue exclusion

#### **4.3. Western blot analysis**

Transfected CHO cells were collected and whole cell lysates were prepared in Complete Buffer (Roche Applied Science) according to the manufacturer's instructions. Total protein was estimated according to Bradford [12]. Protein (100 g) was subjected to 10-20% SDS-PAGE (Invitrogen). Proteins were transferred to nitrocellulose membrane and probed with PRLR isoform specific antibodies (10 g/ml for LF, 6 g/ml for SF1a or SF1b). Reactivity was detected using ECL Plus (GE Healthcare Life Science, Pittsburgh, PA). Molecular size determinations were made using BenchMark Protein Ladder (Invitrogen).

#### **4.4. Fluorescent immunocytochemistry**

CHO cells were plated on 8-well glass chamber slides (Nunc) and transfected as above. After blocking with 5% normal goat serum (Jackson Laboratories, Bar Harbor, ME) prepared in PBS-0.1% Triton, slides were incubated with the PRLR isoform specific monoclonal antibodies (10 g/ml for LF, 6 g/ml for SF1a or SF1b) overnight at 4o C. In all cases no primary antibody served as the negative control. Slides were washed four times with PBS-0.1% Triton followed by incubation for 1 hr with either red fluorescent tagged goat antimouse secondary antibody for the PRLR-LF or red fluorescent tagged goat anti-rabbit secondary antibody for the PRLR-SF (AlexaFluor 594, 1:500, Invitrogen) in the dark. After extensive washing with PBS containing Triton, slides were mounted with Prolong Gold antifade reagent with DAPI (Invitrogen). The fluorescent staining pattern of the receptor isoforms was evaluated using an Olympus BX40 fluorescence microscope (Olympus America, Center Valley, PA).

Prolactin Receptor Isoforms in Human Breast Cancer 199

Nine samples of paraffin-embedded breast tumor tissue were stained and analyzed as

Because serial sections for the breast specimens were used, the same region of each tissue could be measured for fluorescence intensity using Adobe Photoshop (Adobe Systems Inc., Beaverton, OR). Nearly every cell in positive samples showed some level of PRLR isoform expression; as a result, red fluorescence intensity was used to compare levels of isoform expression between samples. In order to do this, the same fluorescent areas were selected from each serial section using the lasso and rectangular marquee tools. Selected sections were analyzed using the histogram function through the red channel, which gave the mean red intensity of the selected section. Photoshop assigns intensity values between 0 and 255 to each pixel in the selected area and then averages these intensities. The distribution of these means was analyzed and used to divide samples into four intensity classes: negative (less than 10 intensity), low (between 11 and 30 intensity), medium (between 31 and 50

Antibodies against the various PRLR isoforms were directly conjugated using the ProbeMaker protocol as described by the manufacturer (OLINK BioScience, Upsala, Sweden). Briefly, 2 l ProbeMaker conjugation buffer was added to 20 g of each antibody (starting antibody concentration, 1 mg/ml) prior to addition of the antibody to the lyophilized oligonucleotides (PLUS and MINUS probes). Incubation with oligonucleotides proceeded overnight at room temperature, followed by addition of 2 l of ProbeMaker Stop Reagent and incubation for 30 min at room temperature to conclude the labeling reactions. Unreacted oligonucleotides were rendered inactive during this step, preventing spurious signals from being generated. For storage of the antibody-oligonucleotide conjugates, 24 l ProbeMaker storage buffer was added. The final concentration of the antibody-

Cultured cells were plated onto 8-well chamber slides (Millipore) in media for one day prior to stimulation as described above. Cells were stimulated for 0, 5, 10, or 60 min with PRL, washed twice in PBS, then fixed in 4% paraformaldehyde for 10 min. Fixed cells were

On the day of the experiment, slides were thawed at room temperature and rehydrated in PBS for 10 min at room temperature. All steps of the Duolink protocol were performed as open droplet reactions on the chamber slides, using 30 l volume per chamber to ensure complete coverage of the cells. Cells were then permeabilized in PBS+0.1% Triton X-100 for 10 min at room temperature, washed twice in PBS, and blocked in 1X Duolink Blocking

above.

**4.6. Measurement of fluorescence intensity** 

intensity), and high (greater than 51 intensity).

**4.8.** *In situ* **proximity ligation assay** 

**4.7. Generation of ProbeMaker probes for Duolink** 

oligonucleotide conjugates were ~0.4 mg/ml and stored at 4o C until use.

subsequently washed twice with PBS, dried and frozen at -20o C.

#### **4.5. Fluorescent immunohistochemistry**

Fresh breast samples were supplied by either the Cooperative Human Tissue Network, a NCI supported resource that supplies human biospecimens to IRB approved researchers, or from patient samples collected in accordance with the guidelines of the National Cancer Institute Review Board, protocol 02-C-0144. Breast tissue was obtained from 15 premenopausal reduction mammoplasty patients; each sample was determined free from hyperplastic growth by a pathologist. Samples were fixed in 10% normal buffered formalin, embedded, cut into four micron sections, deparaffinized, and stained as above. For dual labeling studies, sections were incubated overnight at 4o C with LF (10 g/ml) and either SF1a or SF1b (6 g/ml) monoclonal antibodies. Red fluorescent anti-mouse secondary antibody (AlexaFluor 594, 1:500) was used for LF; green fluorescent anti-rabbit secondary antibody (AlexaFluor 588, 1:500) was used for SF1a and SF1b.

An additional eight samples were also snap-frozen and stored at -80o C for OCT embedding and sectioning. Frozen tissue sections were thawed for 30 to 60 sec and fixed in ice-cold methanol:acetone (1:1) for 10 min. After washing twice with PBS, sections were blocked for 30 min with 10% normal goat serum in IF buffer (PBS containing 0.05 mg/ml sodium azide, 0.1 mg/ml BSA, 0.02% Triton X, and 0.05% Tween 20). Sections were incubated with either LF (10 g/ml) and SF1a or SF1b (6 g/ml) PRLR isoform specific monoclonal antibodies overnight at 4o C. Sections were extensively washed with PBS, then incubated with red fluorescent anti-mouse secondary antibody (AlexaFluor 594, 1:500) for LF and green fluorescent anti-rabbit secondary antibody (AlexaFluor 588, 1:500) for SF1a and SF1b. Sections were washed again and mounted with Prolong Gold antifade reagent with DAPI.

The fluorescent staining patterns of the PRLR isoforms were assessed using an Olympus BX40 fluorescent microscope. For both paraffin-embedded and frozen tissue sections, photographs were immediately taken under the violet, green, and blue channels to detect DAPI, red, and green fluorescence, respectively. Images were merged in order to observe PRLR isoform localization patterns.

Nine samples of paraffin-embedded breast tumor tissue were stained and analyzed as above.

#### **4.6. Measurement of fluorescence intensity**

198 Prolactin

**4.4. Fluorescent immunocytochemistry** 

America, Center Valley, PA).

PRLR isoform localization patterns.

**4.5. Fluorescent immunohistochemistry** 

antibody (AlexaFluor 588, 1:500) was used for SF1a and SF1b.

CHO cells were plated on 8-well glass chamber slides (Nunc) and transfected as above. After blocking with 5% normal goat serum (Jackson Laboratories, Bar Harbor, ME) prepared in PBS-0.1% Triton, slides were incubated with the PRLR isoform specific monoclonal antibodies (10 g/ml for LF, 6 g/ml for SF1a or SF1b) overnight at 4o C. In all cases no primary antibody served as the negative control. Slides were washed four times with PBS-0.1% Triton followed by incubation for 1 hr with either red fluorescent tagged goat antimouse secondary antibody for the PRLR-LF or red fluorescent tagged goat anti-rabbit secondary antibody for the PRLR-SF (AlexaFluor 594, 1:500, Invitrogen) in the dark. After extensive washing with PBS containing Triton, slides were mounted with Prolong Gold antifade reagent with DAPI (Invitrogen). The fluorescent staining pattern of the receptor isoforms was evaluated using an Olympus BX40 fluorescence microscope (Olympus

Fresh breast samples were supplied by either the Cooperative Human Tissue Network, a NCI supported resource that supplies human biospecimens to IRB approved researchers, or from patient samples collected in accordance with the guidelines of the National Cancer Institute Review Board, protocol 02-C-0144. Breast tissue was obtained from 15 premenopausal reduction mammoplasty patients; each sample was determined free from hyperplastic growth by a pathologist. Samples were fixed in 10% normal buffered formalin, embedded, cut into four micron sections, deparaffinized, and stained as above. For dual labeling studies, sections were incubated overnight at 4o C with LF (10 g/ml) and either SF1a or SF1b (6 g/ml) monoclonal antibodies. Red fluorescent anti-mouse secondary antibody (AlexaFluor 594, 1:500) was used for LF; green fluorescent anti-rabbit secondary

An additional eight samples were also snap-frozen and stored at -80o C for OCT embedding and sectioning. Frozen tissue sections were thawed for 30 to 60 sec and fixed in ice-cold methanol:acetone (1:1) for 10 min. After washing twice with PBS, sections were blocked for 30 min with 10% normal goat serum in IF buffer (PBS containing 0.05 mg/ml sodium azide, 0.1 mg/ml BSA, 0.02% Triton X, and 0.05% Tween 20). Sections were incubated with either LF (10 g/ml) and SF1a or SF1b (6 g/ml) PRLR isoform specific monoclonal antibodies overnight at 4o C. Sections were extensively washed with PBS, then incubated with red fluorescent anti-mouse secondary antibody (AlexaFluor 594, 1:500) for LF and green fluorescent anti-rabbit secondary antibody (AlexaFluor 588, 1:500) for SF1a and SF1b. Sections were washed again and mounted with Prolong Gold antifade reagent with DAPI.

The fluorescent staining patterns of the PRLR isoforms were assessed using an Olympus BX40 fluorescent microscope. For both paraffin-embedded and frozen tissue sections, photographs were immediately taken under the violet, green, and blue channels to detect DAPI, red, and green fluorescence, respectively. Images were merged in order to observe Because serial sections for the breast specimens were used, the same region of each tissue could be measured for fluorescence intensity using Adobe Photoshop (Adobe Systems Inc., Beaverton, OR). Nearly every cell in positive samples showed some level of PRLR isoform expression; as a result, red fluorescence intensity was used to compare levels of isoform expression between samples. In order to do this, the same fluorescent areas were selected from each serial section using the lasso and rectangular marquee tools. Selected sections were analyzed using the histogram function through the red channel, which gave the mean red intensity of the selected section. Photoshop assigns intensity values between 0 and 255 to each pixel in the selected area and then averages these intensities. The distribution of these means was analyzed and used to divide samples into four intensity classes: negative (less than 10 intensity), low (between 11 and 30 intensity), medium (between 31 and 50 intensity), and high (greater than 51 intensity).

#### **4.7. Generation of ProbeMaker probes for Duolink**

Antibodies against the various PRLR isoforms were directly conjugated using the ProbeMaker protocol as described by the manufacturer (OLINK BioScience, Upsala, Sweden). Briefly, 2 l ProbeMaker conjugation buffer was added to 20 g of each antibody (starting antibody concentration, 1 mg/ml) prior to addition of the antibody to the lyophilized oligonucleotides (PLUS and MINUS probes). Incubation with oligonucleotides proceeded overnight at room temperature, followed by addition of 2 l of ProbeMaker Stop Reagent and incubation for 30 min at room temperature to conclude the labeling reactions. Unreacted oligonucleotides were rendered inactive during this step, preventing spurious signals from being generated. For storage of the antibody-oligonucleotide conjugates, 24 l ProbeMaker storage buffer was added. The final concentration of the antibodyoligonucleotide conjugates were ~0.4 mg/ml and stored at 4o C until use.

#### **4.8.** *In situ* **proximity ligation assay**

Cultured cells were plated onto 8-well chamber slides (Millipore) in media for one day prior to stimulation as described above. Cells were stimulated for 0, 5, 10, or 60 min with PRL, washed twice in PBS, then fixed in 4% paraformaldehyde for 10 min. Fixed cells were subsequently washed twice with PBS, dried and frozen at -20o C.

On the day of the experiment, slides were thawed at room temperature and rehydrated in PBS for 10 min at room temperature. All steps of the Duolink protocol were performed as open droplet reactions on the chamber slides, using 30 l volume per chamber to ensure complete coverage of the cells. Cells were then permeabilized in PBS+0.1% Triton X-100 for 10 min at room temperature, washed twice in PBS, and blocked in 1X Duolink Blocking solution for 30 min at 37o C in a humidity chamber to prevent evaporation. PRLR primary antibody-oligonucleotide conjugates were diluted in PLA Probe Diluent to 5 g/ml, and applied to slides overnight at 4o C in a cooled humidity chamber. On the second day, slides were washed 3 times in 1X Duolink Wash Buffer A to remove excess antibodyoligonucleotide conjugates. Bound antibody-oligonucleotide conjugates were ligated together for 30 min at 37o C according to the manufacturer's instructions. Subsequently, ligated templates were amplified for 2 hr at 37o C according to the manufacturer's protocol, washed twice for 10 min in Duolink 1X Wash Buffer B, and once for 1 min in 0.1X Wash Buffer B prior to mounting with Duolink Fluorescence mounting media (which also contains DAPI for nuclear counterstaining) and observation under a fluorescent microscope.

Prolactin Receptor Isoforms in Human Breast Cancer 201

**Figure 1.** Characterization of PRLR isoform antibodies. **A**. Western blot analysis indicating specificity of monoclonal antibodies. CHO cells were transiently transfected with isoform specific PRLR cDNA as described. Cell lysates were prepared and proteins separated by PAGE. Each isoform specific lysate was probed with each isoform specific antibody. Western blot analysis was performed twice with separate lysates. Data shown are a representative autoradiogram. Molecular weights are marked as indicated: LF = 93kDa, SF1a = 55kDa, SF1b = 43kDa. **B**. Fluorescent immunocytochemical analysis. CHO cells were transiently transfected with isoform specific PRLR cDNA as described. Specific staining was observed. The negative control lacks primary antibody. Data shown are representative of triplicate experiments.

Magnification 20X.

Image acquisition was performed using a Zeiss Axioscope M1 under 40X magnification and an AxioCam HR. Image analysis was performed using the Duolink ImageTool software according to the instructions provided by OLINK.
