A hormone receptor-positive, HER2/Neu-negative patient who progressed on endocrine hormone therapy participated in the present study. The study was approved by Institutional Ethics Committee III of the Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), and registered with the clinical trial registry of India (CTRI; registration no. CTRI/2017/11/010553; registered on 17/11/2017). The patient's sample was deposited in a biobank at the time of biopsy in a magnetic-activated cell sorting tissue storage solution (Miltenyi Biotec, Inc.). Half of the tissues were used for the development of PDOX and the other half was used for the establishment of primary cultures from patients' tumors. These dissected tissues were fixed in formalin solution.

The animal procedures were approved by and performed in compliance with the guidelines of the Institutional Animal Care and Use Committee of the National Centre for Cell Science (Pune, India). The breast tumor tissues were minced, collected in 1X Dulbecco's PBS and centrifuged at 123 × g for 10 min at room temperature. Non-dissociated tissue fragments (5–10 pieces of <1 mm in size) were administered orthotopically along with 1:1 Matrigel^® (Corning, Inc.) into the right inguinal mammary fat pad of a single female NOD/SCID mouse (age, 8 weeks; body weight, 19 g), which was obtained from the institutional animal facility, National Centre for Cell Science (Pune, India). The mouse was maintained at the in-house pathogen-free facility with ad libitum access to sterile food and chlorinated sterile water at 22±1°C temperature with 55±2% humidity and a 12-h light/dark cycle, and the tumor gradually developed. Following the generation of palpable tumors (approximately within 180 days from date of implantation), the tumor volume was measured using a digital caliper twice a week. The mice were sacrificed when the tumors attained a volume of >2,000 mm³ and a diameter of >20 mm. The mice were sacrificed using CO₂ asphyxiation by maintaining the CO₂ flow rate at 30–70% of the cage volume per minute to minimize sudden distress and pain. Death of the mice was verified by observing the cessation of breath and heartbeat, as well as areflexia as per the guidelines of the Institutional Animal Care and Use Committee of the National Centre for Cell Science, India. The tumors were excised and the weights were measured. A part of the tumor tissue was again implanted orthotopically into the right mammary fat pad of the next set of female NOD/SCID mice (n=1-3) for the development of subsequent generations of PDOXs.

Formalin-fixed and paraffin-embedded tissue sections with 5-µm thickness were stained with H&E. Patients' and PDOX tumor sections were deparaffinized using 3 rounds of fixation in xylene and rehydration in descending grades of ethanol (100, 95 and 75%), followed by distilled water at room temperature. The tissue sections were incubated with a hematoxylin solution for 8 min and washed with water. The sections were soaked in 70% ethanol solution containing 1% HCl and washed again. Subsequently, they were stained with eosin for 5 min and rinsed with absolute alcohol and xylene for 5 min each at room temperature. The images were captured using a bright-field microscope and analyzed by an expert histopathologist.

The tumor tissues from various generations were lysed using RIPA lysis buffer. Following centrifugation, the cleared supernatant was used to measure the total proteins using Bradford's method. The lysates containing an equal amount of total protein (30 µg) were resolved by 10 and 12.5% SDS-PAGE. Protein was transferred onto PVDF membrane (Bio-Rad Laboratories, Inc.) and further analyzed by western blot analysis. The expression levels of ERα, PR, HER2, E-cadherin, N-cadherin and vimentin in tumor tissues derived from different generations of PDOX models were analyzed using their respective primary antibodies, namely anti-ERα (cat. no. ab32063; 1:1,000 dilution; Abcam), anti-PR (cat. no. 8757; 1:1,000 dilution; Cell Signaling Technology, Inc.), anti-HER2 (cat. no. ab2428; 1:1,000 dilution; Abcam), anti-E-cadherin (cat. no. ab1416; 1:1,000 dilution; Abcam), anti-N-cadherin (cat. no. ab18203; 1:1,000 dilution; Abcam) and anti-vimentin (cat. no. sc-7558; Santa Cruz Biotechnology, Inc.; 1:1,000 dilution), followed by incubation for 1 h at room temperature with specific secondary antibodies, including goat anti-rabbit IgG HRP (cat no. 114038001A; Genei Laboratories Pvt. Ltd.), rabbit anti-mouse IgG HRP (cat no. 114058001A; Genei Laboratories Pvt. Ltd.) and rabbit anti-goat IgG HRP (cat no. 114048001A; Genei Laboratories Pvt. Ltd.) at 1:2,000 dilution. All the blots were visualized using Clarity Western ECL (Bio-Rad Laboratories, Inc.) reagent.

The primary cultures were established as previously described with minor modifications (14). The clinical specimens were aseptically transferred into DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 4% penicillin and streptomycin solution. The tissue specimens were washed twice with 1X PBS containing an antibiotic solution. The tissue specimens were cut into fine pieces and incubated in 0.15% collagenase II and collagenase IV (Gibco; Thermo Fisher Scientific, Inc.) solution in a water bath at 37°C for 2 h for proper enzymatic digestion. Following incubation, the tissue specimens were centrifuged at 123 × g for 10 min at room temperature. The pellets were resuspended in DMEM supplemented with 20% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 25 µl hydrocortisone (Calbiochem), 10 µl insulin (Gibco; Thermo Fisher Scientific, Inc.), 5 µl epidermal growth factor (Peprotech) and a solution containing 2% penicillin and streptomycin (Gibco; Thermo Fisher Scientific, Inc.). The cells were grown in a 95% humidified incubator with 5% CO₂ at 37°C. When the cultures were established, the amount of FBS was reduced to 10% to maintain the selective growth of cancer epithelial cells.

Primary cultures were characterized by immunofluorescence as previously described (15). The primary culture cells were grown on coverslips. When the cells were confluent, they were fixed in 2% paraformaldehyde for 20 min, quenched with 100 mM glycine for 10 min, permeabilized with 0.1% Triton-X100 for 10 min and incubated with 10% FBS in PBS to block non-specific binding for 1 h at room temperature. The cells were incubated overnight at 4°C with primary antibodies to the following proteins: estrogen receptor (ER)-α (cat. no. ab32063; Abcam), progesterone receptor (PR) (cat. no. ab8757; Cell Signaling Technology, Inc.), HER2 (cat. no. ab2428; Abcam), E-cadherin (cat. no. sc-7870; Santa Cruz Biotechnology, Inc.), N-cadherin (ab18203; Abcam), vimentin (sc-7558; Santa Cruz Biotechnology, Inc.), Ki-67 (sc-23900; Santa Cruz Biotechnology, Inc.), α-smooth muscle actin (SMA; ab5694; Abcam) and pan-cytokeratin (cat. no. C5992; Sigma-Aldrich; Merck Millipore) at 1:100 dilution. Fluorescent-labeled secondary antibodies such as goat anti-mouse (cat. no. AP124C), donkey anti-rabbit (cat. no. AP182C) and donkey anti-goat (cat. no. AP180C) Cy3 antibodies were added to cells at 1:200 dilution and incubated for 1 h at room temperature. The cells were visualized using a confocal microscope (Leica Microsystems).

The data were analyzed and graphs were prepared using SigmaPlot version 10.0 (Systat software).

A 51-year-old post-menopausal female with no comorbidities was referred to the Tata Memorial Hospital (Mumbai, India) in October 2013 with a 5-cm lump in the left breast and a 2-cm lymph node (left axilla). The case had been determined to be positive for the presence of a tumor based on a computerized tomography scan. The patient had previously received four cycles of cyclophosphamide, adriamycin and 5-fluorouracil prior to registering at our center. A bone scan revealed the presence of asymptomatic oligometastatic disease with suspected osteoblastic lesions in the ribs. The patient provided consent for further clinical treatment options and agreed to the treatment with radical curative intent by simple mastectomy. Subsequently, the patient received localized radiation therapy (RT) at the site of excision, taxane-based chemotherapy and aromatase inhibitors, which were completed in April 2014. The patient was disease-free for the next two years and presented with a recurring disease in the liver in October 2016. Therapy with tamoxifen and paclitaxel was initiated, along with zoledronic acid. The patients also received palliative RT to the D4-D6 region to alleviate symptomatic pain. The patient remained stable on tamoxifen in the absence of disease progression for 1 year. In 2017, following the appearance of new metastatic nodules in the liver, the patient received therapy containing exemestane. The patient remained stable on exemestane for another year prior to the appearance of new nodules in the lung, which were consistent with the presence of lung metastatic disease. The patient provided consent to being recruited for the present study in 2018. A biopsy was performed in 2–3 tumor cores from the patient's liver using ultrasound guidance. The tissues were added to a biobank for subsequent processing. The patient was subsequently treated with capecitabine therapy along with concurrent palliative RT. The patient progressed within two months and received therapy based on a protocol of gemcitabine and carboplatin. The patient responded to therapy for six months and had stable disease. Subsequently, disease progression was observed in early 2019. The patient received letrozole and zoledronic acid followed by palliative RT. The patient continued receiving therapy containing letrozole and zoledronic acid in 2019, exhibited stable disease and was alive at the last follow-up in April 2022.

The histopathological examination of the tumor resected in 2013 identified it as an infiltrating duct carcinoma of the cribriform type with a high nuclear grade (grade III) and necrosis. The tumor exhibited a focal micropapillary pattern and extracellular mucin with a modified Bloom Richardson Score of 3+3+2=8 in the absence of Paget's disease (Fig. 1A). The tumor exhibited positive staining for ER (all-red score, 8) and PR (all-red score, 8), as determined by immunohistochemistry (IHC) (Fig. 1A-C) and negative for HER2 expression on the Ventana system (score +1; data not shown). Histopathological assessment of lymph nodes assessed by axillary clearance during surgery revealed the presence of cancer cells in 10/20 lymph nodes assessed. Histopathological re-examination of the tumor biopsied from the patient's liver at progression in January 2018 revealed consistency with the resected tumor prior to five years. The tumor also demonstrated positive staining for ER (all-red score, 8) and PR (all-red score, 8; Fig. 1D-F) and negative for HER2 expression based on the Ventana system (score +1; data not shown).

Subcutaneous tumor PDX models are standard pre-clinical models that are widely used to study treatment response. However, they also exhibit low clinical relevance and biological features to patients' tumors as compared to orthotopic PDXs. Furthermore, other major limitations of the subcutaneous models include a lower potential to metastasize and failure to accurately mimic the tumor microenvironment and imitate a patient's clinical treatment response. In contrast to these observations, PDOXs may accurately and precisely epitomize the clinical behavior and treatment response of a patient's unique cancer (11).

In the present study, a breast PDOX model was developed using hormone-resistant-breast cancer by orthotopically implanting tumor fragments into the mammary fat pad of female NOD/SCID mice. The tissues were mixed with Matrigel to mimic the organ microenvironment prior to implantation. The rate of successful engraftment from the patient tumors to the NOD/SCID mice was estimated to be 20% (3 PDOXs were established out of 15 samples implanted; data not shown), which was consistent with previously reported studies (16,17). However, the in vivo passaging rate was considerably higher (~90%) following the establishment of PDOXs. Following 70 days of implantation, the tumor was grown to a palpable size (Generation 1; G1). The tumor was grown to the endpoint volume, (~2,000 mm³) within 90 days after the observation of the palpable tumors. Serial passaging of established PDOXs was performed in the next cohort of immunocompromised mice to establish G2 PDOXs. PDOXs exhibited an unstable growth pattern. The time required to attain palpable tumors (tumor take) for G2 was 60 days, whereas that for G3 was ~40 days. The time required to reach the tumor burden from the stage of palpable tumors for G2 was 60 days, while that for G3 (to reach the same volume as G1) was 50 days (Fig. 2A and B). The time periods required to develop palpable tumors and to reach the tumor burden for G4 were considerably decreased compared with those of the G1-G3 generations of PDOXs. This is likely due to the corresponding patient having undergone chemotherapy prior to surgery. Therefore, PDOXs exhibited signs of chemotherapy-induced differentiation and slow tumor-cell proliferation in the lower passage and aggressive growth in advanced cohorts (Fig. 2A and B).

The tissue sections derived from primary tumors and PDOXs were stained with H&E to assess their histological features and determine growth stability and tissue morphology. The histological reports of primary tumors and PDOXs revealed similar grade histology represented by minimal to maximal nuclear polymorphism and vessel formation, numerous mitotic activities with occasional abnormal mitoses including atypical mitotic figures and areas of necrosis (Figs. 1A and 3A-D, Table I). Mitotic activity and necrosis were significantly increased from G1 to G4 (Fig. 3A-D, Table I). The increasing trend was in parallel with the unstable growth patterns observed in the PDOXs (Fig. 2B, Table I). The tumor tissues of different passages of PDOXs were subjected to immunofluorescence and western blot analyses to further characterize the expression of subtype- and epithelial-to-mesenchymal transition (EMT)-specific markers. The results indicated that the expression levels of breast cancer-specific markers, such as ERα and PR, as well as those of the EMT markers, such as E-cadherin, N-cadherin and vimentin, were similar in the different passages of PDOXs (Fig. 4A-C). To further characterize their subtype, the primary cultures were established and immunofluorescence analysis was performed to examine the expression levels of ERα, PR and HER2, and those of the epithelial- and mesenchymal-specific markers, such as E-cadherin, N-cadherin, vimentin and α-SMA. The expression levels of subtype-specific markers, such as ERα, PR and Ki-67, were observed in the primary culture derived from G1 PDOXs and were mostly retained in primary culture of G4 PDOXs. The epithelial and mesenchymal markers, such as E-cadherin, N-cadherin, vimentin and pan-cytokeratin, were also similar between primary cultures of G1 and G4 PDOXs (Fig. 5A and B).

From these observations, it was suggested that the histological and molecular features of PDOXs were mostly retained in different passages of PDOXs. All these data demonstrated the successful development of the hormone therapy-resistant breast cancer PDOX models from an Indian patient. These models were extended to four generations and their luminol-positive features were characterized compared with those of the patient's tissues. These models will be used for drug screening and the development of therapeutically relevant biomarkers using proteomic and genomic-based approaches. All of these works are in progress and will be published soon in a separate article.