Characterizing a murine breast cancer mouse model reveals chromosomal abnormalities in structure and number of single‐cell clones and the presence of rare cancer stem cell‐like phenotypes

Abstract

Realistic models for cancer research representing disease progression that commensurately respond to therapeutics consistent with clinical observation are the holy grail for pre‐clinical research and screening. Although such an ideal is elusive, well‐characterized in vivo models facilitate our understanding of disease, progression, and therapeutic opportunities. Here, we characterize a commonly used syngeneic BALB/c mouse model of triple negative breast cancer (4T1) after establishing tumors in their flanks. Tumors developed at the subcutaneous injection site for all experimental mice and their volumes were monitored. We quantified a rare subset of breast cancer stem‐like cells (CSCs), classified as CD44⁺/CD24⁻ phenotypes in in vitro and ex vivo cell populations. Chromosome numbers in ex vivo metaphase cells were greater than cells cultured in vitro (89.4 ± 3.4, range of 70–132 and 82.6 ± 1.1, range of 70–128; respectively). Further, we observed different types of chromosome aberrations, including gap, deletion, exchange, interstitial deletion, terminal deletion, ring, dicentric, and Robertsonian translocations. For both sources of cells, the number of aberrations was dominated by deletions, terminal deletions, and Robertsonian translocations. Ex vivo cells exhibited greater prevalence of deletions and terminal deletions, whereas in vitro cells displayed more ring aberrations and Robertsonian translocations. In conclusion, we successfully characterized cancer cells from a syngeneic mouse model of breast cancer in terms of rare CSC proportion and a variety of chromosomal aberrations, which is useful for understanding tumor traits associated with cancer development and therapeutic action. The data act as a valuable resource for other studies using the 4T1 BALB/c model.

Characterizing a murine breast cancer mouse model reveals chromosomal abnormalities in structure and number of single‐cell clones and the presence of rare cancer stem cell‐like phenotypes.

1. INTRODUCTION

Breast cancer is a substantial public health risk. Pre‐clinical cancer research attempts to simulate realistic patient and clinical scenarios. The murine syngeneic model of breast cancer with 4T1 cancer cells is a valuable representative of human breast cancer based on traits such as poor prognosis, aggressive invasion and metastasis, ¹ and downregulated expression of progesterone (PR), estrogen (ER), and human epidermal growth factor receptors (HER‐2) ² analogous to a triple negative phenotype.

Chromosome aberrations are associated with cancer progression. ³ A variety of biological processes, including gene dysregulation and genomic instability in cells, have been linked to chromosomal abnormalities. ⁴ , ⁵ The chromosomal abnormality types detected in solid tumors have been linked to other cellular alterations, including polyploidy and aneuploidy. ⁶ An increase in chromosome number can result in activated oncogenes being amplified and lead to treatment resistance. For example, after treatment with endocrine therapy, amplification of the androgen receptor gene (AR) promotes prostate cancer cell survival. ⁷

Another negative prognostic factor is the distribution of breast cancer stem‐like cells (CSCs). CSCs have greater resistance to radiation and chemotherapy and are instrumental in repopulating heterogeneous tumor and promoting metastasis. ⁸ , ⁹ , ¹⁰ , ¹¹ Due to their robust self‐renewal and differentiation capacities, CD44⁺/CD24⁻ CSCs have been associated with triple negative breast cancer (TNBC) relapse, ¹² aggressiveness, and negative prognosis. ¹³ These CSCs have the potential for innate or acquired chemoresistance via a variety of pathways. ¹⁴ For example, CD44⁺/CD24⁻ CSCs exhibit resistance to doxorubicin and greater proliferation and differentiation. ¹⁵ In addition, CSCs may play an important role in resistance to radiotherapy (RT) by overcoming radiobiological effects by prospectively utilizing radioresistant Notch‐1 and HH signaling pathways. ¹³ , ¹⁶ They subsequently undergo symmetrical CSC division, increasing proportions of breast CD44⁺/CD24⁻ CSCs shortly after radiation treatment. ¹³ Radioresistant capacity of a tumor has thus been associated with the presentation of CD44⁺/CD24⁻CSCs. ¹⁴

Currently, the development of cancer in syngeneic mouse models and the prognostic factors reported for TNBC have not been well characterized. Here, we describe and characterize the subcutaneous 4T1 murine breast cancer cell, syngeneic BALB/c mouse model. This model provides valuable insight into both changes in chromosomal abnormalities in tumor cells and the distribution of cancer stem cell subpopulations that exist in the tumor. This provides an improved foundation for understanding TNBC tumor development. With greater knowledge of the natural development and changes in preclinical models of breast cancer, more effective treatments can be developed.

2. METHODOLOGY

2.1. 4T1 cells and culture

The 4T1 mouse breast cancer cell line was purchased from the American Type Culture Collection (ATCC^R CRL‐2539) (Manassas, Virginia, USA). Cells were grown in 25 cm² flasks containing Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, New York, USA) supplemented with 10% FBS (Gibco, NY, USA), 2 mmol/L‐glutamine, 100 U/mL penicillin–streptomycin antibiotic mixture (Gibco) at 37°C, and 5% CO₂ in a humidified incubator. The adherent 4T1 cells were cultured until reaching 80% confluence. Cells were then detached with Trypsin EDTA 0.25% (Gibco) for 5 min in a 37°C incubator. Stored cells were frozen in a medium containing RPMI 1640, 10% FBS, and 10% dimethyl sulfoxide (DMSO) (Sigma‐Aldrich, Missouri, USA) for 1 week in an isopropanol box at –80°C and subsequently stored in liquid nitrogen until use.

2.2. 4T1 BALB/c mouse model of breast cancer

BALB/c female mice were bred and housed in the Core Animal Facility, University of South Australia, Adelaide, Australia. All work conducted here was approved by the University of South Australia Animal Ethics Committee (U49‐19 and U34‐21). The 4T1 cells were cultured until reaching 80%–90% confluence, then trypsinized with Trypsin–EDTA 0.25%. Cells were suspended with medium and washed with sterile PBS (Gibco) following centrifugation at 250 g for 5 min to remove EDTA and trypsin.

To establish the mouse model, hair was shaved on the left flank of 4–6‐week‐old female BALB/c mice, and 5 × 10⁵ 4T1 cells in 0.2 mL of sterile PBS were inoculated subcutaneously. Mice were monitored every day, and tumor sizes at the inoculated site were measured with digital calipers twice per week to examine tumor growth once palpable. Tumor volume was calculated as V = (L × W × W)/2 (V, volume; L, length; W, width). During that time, mice were killed if tumors reached 15 mm in any direction (length or width). Mice were humanely killed using isoflurane anesthesia and cervical dislocation at day 30 after tumor inoculation. Tumor tissues were excised and fixed in 10% formalin (Sigma‐Aldrich, Missouri, USA) at 4°C overnight and then preserved in 70% ethanol (Sigma‐Aldrich). Tumor samples from three mice were preserved in Tissue Storage Solution (Miltenyi Biotec, Bergisch Gladbach, Germany) at 4°C for 24 h prior to fresh primary single cell preparation.

2.3. Cell suspension preparation

To prepare cell suspensions, tumor samples from mice were isolated, with weight recorded and stored in MACS Tissue Storage Solution overnight. A detailed protocol for harvesting single‐cell populations derived from murine primary breast tissue has been described elsewhere. ¹⁷ Briefly, mouse breast tumor tissue was finely cut with a sterile scalpel for several minutes. Minced tissues were digested in appropriate amounts of digestion buffer, based on diluting 2.5 mg hyaluronidase (Sigma‐Aldrich) and 15 mg collagenase (Sigma‐Aldrich) in 5 mL DMEM/F12 (Gibco) per 0.5 g of each tumor tissue. This digestion process was performed for 2 h at 37°C and 100 rpm on a shaking incubator. Cell suspensions were then collected by passing the supernatants through a 40‐micron strainer (Sigma‐Aldrich). The cell mixtures were transferred into 15 mL tubes followed by centrifugation at 300 g for 5 min. After centrifugation, cell pellets were resuspended in 4 mL sterile PBS for cell counting via a hemocytometer. These cell suspensions were used for chromosome analysis or flow cytometry analysis for cancer stem cells. Ex vivo cells were prepared from the singe cell suspensions.

2.4. Preparation of the chromosomes of metaphase cells

The in vitro 4T1 cells and ex vivo cells were plated in RPMI 1640 medium (Gibco) supplemented with 10% FBS (Gibco), 2 mmol/L‐glutamine, 100 U/mL penicillin–streptomycin antibiotic mixture (Gibco) and cultured at 37°C and 5% CO₂ in a humidified incubator until reaching 80%–90% confluence and used for examining chromosomal numbers for cells in the metaphase stage. The protocol for chromosome preparation was conducted following the protocol of Farkas et al. ¹⁸ Briefly, once 4T1 cells and ex vivo cells reached 80%–90% confluence in vitro, half of old media was replaced with new culture media. Cells were further cultured for 5 h at 37°C and 5% CO₂ humidified incubation. To arrest cells in the metaphase for chromosomal analysis, 80 μL Colcemid (10 μg/mL, Gibco) was added to 10 mL culture media, and cells were incubated for a further 2 h at 37°C and 5% CO₂ humidified atmosphere prior to harvesting. Cells were then washed with PBS and treated with 0.25% trypsin–EDTA. Cells were suspended in fresh RPMI 1640 media and centrifuged at 250× g for 5 min, and cell pellets were collected. They were then supplied with 0.075 M KCL (Sigma‐Aldrich) and 0.9% sodium citrate (Sigma‐Aldrich), gently flicked and incubated at room temperature for 7 min 30 s. Cell samples were fixed with a cold fixation solution of three parts methanol to one‐part acetic acid. The samples were fixed five times altogether and then incubated in the last fixative in a –20°C freezer overnight. For chromosome analysis, cell suspensions were transferred onto slides and stained with 3% Giemsa (Life Technologies, California, USA) for 5 min at room temperature. All slides were mounted with mounting medium and analyzed with 1000× magnification. Chromosome numbers and aberrations were manually counted. Wang et al. ¹⁹ showed that both cancer‐associated fibroblasts and normal fibroblasts in mice were displayed as normal mouse karyotypes, consisting of a total of 40 chromosomes. The presence of more than 40 chromosomes in ex vivo cells suggest the presence of cancer cells. Only spreads with 70 or greater chromosomes were included in the analysis to avoid the inclusion of normal cells or spreads with debris from other broken cells.

2.5. Stem cell analysis

Stem cell populations were analyzed with flow cytometry based on CD44⁺/CD24⁻expression. Cells were incubated with specific antibodies anti‐CD44‐APC (cat. no.: 103012) and anti‐CD24‐PE (cat. no.: 101807) (Bio Legend, California, USA). 4T1 cells and ex vivo cells were cultured until 80%–90% confluence. One million 4T1 cells or ex vivo cells were suspended in PBS, and anti‐CD44‐APC and anti‐CD24‐PE at 2 μg/mL were added and left for 30 min on ice in the dark. Cells were washed two times with 1 mL sterile PBS followed by centrifugation at 250 g (RCF) for 5 min. After washing, cell pellets were resuspended in 500 μL PBS and measured on a BD LSRFortessa X‐20 flow cytometer (BD Biosciences, Franklin Lakes, New Jersey, USA) and data were analyzed with BD FACSDiva 9.0.1 (BD Biosciences).

2.6. H&E staining

Fresh tumor tissues were collected and immediately transferred into 10% neutral formalin (Sigma‐Aldrich) for overnight fixation in the fridge. The following day, they were washed twice with PBS and supplemented with 70% ethanol (Sigma‐Aldrich). The fixed samples were then processed through an automated tissue processor (Leica ASP300, Leica, Wetzlar, Germany). The tissues went through paraffin embedding processing (Leica EG1160, Germany), and sections were prepared by microtome (Leica RM2155). Sections were placed on glass slides and dried at 40°C overnight before being dewaxed in xylene solution for 2 min, followed by 100%, 70%, 30% ethyl alcohol and H₂O. Finally, sections were stained with hematoxylin and eosin (H&E). Slides were mounted by coverslip and evaluated by bright field with an Olympus IX81 microscope (Olympus, Tokyo, Japan) under 40X magnification with CellSens Microscope Imaging Software (Olympus).

2.7. Statistical analysis

Statistical analyses were conducted with one‐way analysis of variance (ANOVA) and Mann–Whitney tests (GraphPad Prism version 8, USA). Data normality was tested by the Kolmogorov–Smirnov test. For statistical comparison of chromosome aberrations between ex vivo and in vitro grown cells, all deletion aberrations were pooled into one aberration class and the data for all aberration classifications were compared via a Fisher's exact test to accommodate low and zero frequency data for some conditions. A p‐value of <0.05 was considered significant.

3. RESULTS

3.1. Subcutaneous tumor development

Seven female BALB/c mice were injected with 4T1 murine breast cancer cells to develop a syngeneic mouse model. Mice were monitored for weight and tumor growth for 30 days (Figure 1A). Tumors developed in 100% of mice with tumor volumes at day 7 of 61.7, 22.1, 19.8, 63.8, 64.7, 88, 36.1 mm³, respectively, similar to other work. ²⁰ Tumors grew steadily over the 30 days, as shown in Figure 1B. Two mice were humanely killed on days 22 and 28 as their tumor size reached a humane endpoint. At 30 days, all remaining mice were humanely killed with tumor volumes being 1196.71, 1027.29, 838.99, 1071.07, and 891.62 mm³. Figure 1C shows an example of a tumor excised at day 30, and Figure 1D shows an H&E section of that tumor, which revealed mostly viable tumor cells with pleomorphic vesicular nuclei and hyperchromic prominent nucleoli. ²⁰

FIGURE 1.

A 4T1 BALB/c mouse model of breast cancer. The left flanks of mice were injected with 4T1 cells subcutaneously. (A) A representative picture of an injected mouse is shown with a white arrow indicating the site of primary tumor growth. (B) The tumor volume was evaluated twice per week. (C) A representative excised tumor. (D) H&E staining of primary tumor tissue at the end point of tumor development with pleomorphic vesicular nuclei (green arrows) and hyperchrome prominent nucleoli (yellow arrows) indicated (40× magnification, and the scale bar represents 20 μm).

3.2. Assessment of chromosome aberrations in 4T1 cells

Chromosome aberrations were analyzed by following the previous reports of Kocsis et al. ²¹ One hundred single metaphase cells from in vitro culture were compared to 23 single ex vivo cells. Chromosome number data from in vitro cells were normally distributed and that of ex vivo cells were not normally distributed (Kolmogorov–Smirnov test; K‐S distance (100) = 0.135 and K–S distance (23) = 0.114, and p > 0.1 and p < 0.0001, for in vitro and ex vivo samples, respectively). Although the difference was not significant according to a p = 0.05 threshold (Mann–Whitney two‐tailed test, p values = 0.0587), ex vivo cells showed more chromosomes than in vitro cells shown in Figure 2A,B.

FIGURE 2.

Chromosome counts of metaphase cells. (A) Chromosome number of murine 4T1 breast cancer cells prepared ex vivo (23 cells) and in vitro (100 cells). Boxes represent data range from 1st quartile to 3rd quartile. Whiskers represent the range of chromosomes from minimum to the maximum. (B) Descriptive statistics and Mann–Whitney two‐tailed test results. (C) Distribution of chromosome numbers in ex vivo and in vitro cells.

The mean chromosome number of mouse breast cancer cells prepared ex vivo and in vitro was 89.4 ± 3.4 (SEM) (range of 70–132 chromosomes) in 23 metaphase spreads and 82.6 ± 1.1 (SEM) (range of 70–128 chromosomes) in 100 metaphase spreads, according to our findings (Figure 2A). Histograms showing the relative frequency distribution and range of data are given in Figure 2C.

Next, we examined structural chromosomal aberrations and observed different types of chromosome aberrations including gap, deletion, exchange, interstitial deletion, terminal deletion, ring, dicentric, Robertsonian translocation (Figure S1). Table 1 shows that ex vivo cells exhibited a greater proportion of cells with deletions and terminal deletions compared to in vitro cells. In contrast, in vitro cells displayed more cells with ring aberrations and Robertson translocations than ex vivo cells. The proportion was calculated by comparing each structural aberration to the total number of aberrations in 23 ex vivo cells and 100 in vitro cells. When normalized to 100 cells, deletion and terminal deletion showed nearly twice the amount in ex vivo cells, with 152.2 and 178.3, respectively, compared to in vitro cells with 84 and 88 deletions (Table 2).

TABLE 1.

The proportion of structural chromosome aberrations.

Types of aberration	Ex vivo cells (n = 23, total aberration = 126)	In vitro cells (n = 100, total aberration = 510)
Gap	4 (3.2%)	12 (2.4%)
Deletion	35 (27.8%)	84 (16.5%)
Exchange	0	1 (0.2%)
Interstitial deletion	0	0
Terminal deletion	41 (32.5%)	88 (17.3%)
Ring	10 (7.9%)	85 (16.7%)
Dicentric	0	7 (1.4%)
Robertsonian translocation	36 (28.6%)	230 (45.1%)
Other	0	3 (0.6%)

TABLE 2.

Number of chromosome aberrations.

Types	Ex vivo cells		In vitro cells
	Normalized to 100 cells	Raw count (23 cells)	Normalized to 100 cells	Raw count (100 cells)
Gap	17.4	4	12.0	12
Deletion	152.2	35	84.0	84
Exchange	0.0	0.0	1.0	1.0
Interstitial deletion	0.0	0.0	0.0	0.0
Terminal deletion	178.3	41	88.0	88
Ring	43.5	10	85.0	85
Dicentric	0.0	0.0	7.0	7.0
Robertsonian translocation	156.5	36.0	230.0	230.0
Other	0.0	0.0	3.0	3.0

3.3. Cancer stem‐like CD44 ⁺/CD24 ⁻ phenotype

CD44 expression and the lack of CD24 presence have been associated with CSC‐like phenotypes in breast cancer. ²² We characterized in vitro 4T1 cells and ex vivo tumor cells by flow cytometry for surface expression of CD44 and CD24. Ex vivo cells were derived from 4T1 cells grown in the flank of mice. Tumor cell suspensions were stained with fluorescent anti‐CD24 and anti‐CD44 markers to determine the percentage of breast CSCs in the related tumor cell suspensions. Flow cytometry analysis was performed on 10 000 in vitro cells and ex vivo cells (Figure 3A) distinguishing CD44⁻/CD24⁺, CD44⁺/CD24⁺, CD44⁺/CD24⁻, and CD44⁻/CD24⁻ subpopulations (Figure 3B). This allows us to compare the distribution of cancer cells with stem‐like phenotype traits both in in vitro cultures and within tumor tissues. The CD44⁺/CD24⁻ CSC phenotype was expressed in in vitro cells and in ex vivo cells of three mice (Figure 3B). The percentage of CD44⁺/CD24⁻ cells ex vivo varied between individual mice, but the average value was consistent with the proportion seen for in vitro cells.

FIGURE 3.

Analysis of breast cancer stem cell‐like phenotypes (CD44⁺/CD24⁻). (A) Flow cytometric analysis of CD24/CD44 expression in ex vivo and in vitro cells. (B) Percentage of four subpopulations in breast cancer ex vivo and in vitro 4T1 cells. Standard deviation is indicated for each respective cell suspension, n = 3.

4. DISCUSSION

Injecting 4T1 cancer cells into the left flanks of adult female BALB/c mice led to tumor formation and growth during the experimental period with a 100% success rate. This has also been reported in other studies involving the inoculation of 4T1cells into mouse fat pads. ²⁰ Cancer cells characteristically, in general, have chromosome numbers differing from normal cells. It has been shown by Stopsack et al. ²³ that tumor cells with aneuploidy are more aggressive and grow much faster. This can alter gene function by activating oncogenes and inactivating tumor suppressors. Chromosomal aberration (CA) frequency has been identified as a specific marker signaling abnormal cell formation that influences tumor evolution and aggressive tumor behavior. ²⁴ Figure 1D depicts a section of primary tumor collected from a mouse using H&E staining. The captured image at high magnification shows tumor cells with pleomorphic vesicular nuclei and hyperchromic nucleoli. These observations are consistent with a tumor developed in the mammary fat pad. ²⁰ These properties promote cancer cells to undergo aneuploidies caused by chromosome aberrations, resulting in changes throughout the entire genome. Chromosomal aberrations have been shown to be associated with tumor stage, relapse, and metastasis in triple‐negative breast cancers. ²⁴ Furthermore, the similarities between tumor genomes show that many advanced cancers have a stable chromosome aberration spectrum in samples collected from primary and recurrent tumors ²⁵ as well as primary and metastatic tumors ²⁶ within individuals. It is less clear though if chromosomal aberration landscape varies with cancer progression. Clonal heterogeneity may occur however in TNBC patients, ²⁷ especially in metastases the development of acquired treatment resistance that will be dependent on intervention. Our data suggest that there are differences between in vitro and ex vivo cells that may indicate early bias toward phenotypes that proliferate in vivo.

Chromosomes play an important role in ensuring that DNA replication is correct and evenly distributed among newly generated cells. Generally, the number of chromosomes from one diploid cell in a mouse is 40 as determined in normal B lymphocyte ²⁸ and embryonic fibroblast cell lines. ²⁹ Embryonic fibroblast cell lines that lack the tumor suppressor protein (Ski) have 33–75 chromosomes. ²⁹ Across four different human breast cancer cell lines MCF7 (clonal ER⁺/HER2⁻), T47D (clonal ER⁺/HER2⁻), BT474 (clonal ER⁺/HER2⁺), and SKBB3 (clonal ER⁻/HER⁺), chromosome numbers ranged from 76–88, 57–66, 65–106, and 76–83 respectively for 19 to 26 cells for each line. ³⁰ Authors also showed that structural aberrations, including deletions, duplications, inversions, translocations, dicentric and ring chromosomes, further shape the genomic landscape of breast tumors. These lesions often originate from double‐strand DNA breaks (DSBs) that are mis‐repaired through non‐homologous end joining (NHEJ) or microhomology‐mediated end joining (MMEJ), particularly in tumors with defective homologous recombination repair (HRR). For instance, BRCA1/2‐deficient contexts lead to chromatid exchanges, deletions, and chromosomal fusions that underlie characteristic “mutational signatures” of homologous recombination deficiency (HRD). The presence of ring and dicentric chromosomes, commonly reported in triple‐negative and HER2‐enriched breast cancers, reflects cycles of breakage–fusion–bridge (BFB) events driven by telomere attrition and loss of telomere‐protective proteins (e.g., TRF2, POT1). These events amplify oncogenes and delete tumor suppressors, reinforcing malignant evolution.

Tumor‐specific cancer stem cell subsets influence preclinical and clinical outcomes by promoting cancer progression and development. We used flow cytometry to investigate the frequency of CD44⁺ and CD24⁻ cells to provide a more precise estimate of these rare cell subsets. ³¹ , ³² CD44⁺/CD24⁺ cells were the most abundant phenotype in ex vivo and in vitro cells. However, these cells were reported by Mylona et al. ³³ to not be associated with invasion and spread. Their study also indicated that CD44⁻/CD24⁺ cells do not hold clinicopathologic significance. CD44⁺/CD24⁻ CSC‐like cells have the ability to initiate tumor growth. ³⁴ Our data are similar to the other published reports of 0.3% CD44⁺/CD24⁻ cells in human breast cancer cell lines MDA‐MB‐468. ³⁴ , ³⁵ CSCs can drive repopulation, spread to distant organs, and become resistant to radiation and chemotherapy. ⁸ , ⁹ , ¹⁰ , ¹¹ Furthermore, the frequency of CD44⁺/CD24⁻ phenotype has been shown to be a dominant factor in aggressive TNBC, correlating with tumor size and metastasis. ¹² , ³⁶

It is possible for breast cancer to develop cells that can survive in circulation and metastatic lesions while also increasing chromosomal heterogeneity and instability. ²⁴ We measured that ex vivo cancer cells have slightly more chromosomes than in vitro cancer cells. These findings suggest that cancer cells grown in a realistic environment in an animal model may have more chromosomes than cancer cells grown in vitro, which would indicate that the tumor experienced aneuploidy and intragenic mutations during tumorigenesis, or that cells with greater aneuploidy dominate in vivo tumor formation. ³⁷ These data indicate that extrapolating in vitro observations to in vivo assumptions may not always be reasonable with respect to chromosomal‐based traits and therapeutic vulnerabilities. Tumors' initiation mechanisms may differ due to differences in the mechanisms that trigger them. The incidence of chromosomal abnormalities has been reported to increase dramatically in in situ breast carcinomas, ³⁸ and a high level of chromosomal aberrations has been linked to poor clinical outcomes in ovarian cancer. ³⁹ As a result, as demonstrated, studying chromosomal abnormalities can provide us with a better picture of their role in cancer evolution. ⁴⁰ In our study, both in vitro and ex vivo murine breast cancer cells were discovered to have structural aberrations.

Tumors with structural chromosome aberrations have instability, which is associated with intra‐tumor heterogeneity, resulting in poorer prognosis and resistance to therapy. All types of solid tumors have chromosomal structural abnormalities. ⁶ , ⁴¹ Cryopreservation also contributes to these anomalies. ⁴² Cryopreservation is a fundamental technique for maintaining long‐term cell line repositories and ensuring experimental reproducibility. The CD44 and CD24 antigens are both surface glycoproteins sensitive to membrane lipid‐phase transitions and oxidative modification. Although CD24 can be shed or enzymatically cleaved, CD44 epitopes can also become masked or denatured by cross‐linking, oxidation, or loss of membrane raft organization during freezing. Consequently, flow‐cytometry detection underestimates true CD44⁺ events because damaged epitopes fail to bind antibodies efficiently. This phenomenon was evident in Le Gallo et al., ⁴³ where the CD44⁻/CD24⁻ and CD44⁺/CD24⁻ subsets both decreased following a single freeze–thaw cycle, despite total tumor‐cell recovery.

Aberrations stem from osmotic and mechanical stresses during freezing/thawing (ice crystal formation, shear, membrane/cytoskeleton disruption, ROS bursts) that can generate chromatid gaps/breaks and subsequent mis‐repair into deletions, translocations, or copy‐number drift. Given that breast cancer cell lines frequently exhibit intrinsic instability, such cryo‐induced drift may confound the interpretation of structural variability (e.g., sub‐clonal rearrangements, aneuploidy changes) and phenotypic assays (e.g., migration, stemness). ⁴⁴

According to our findings, a greater proportion of deletions were there in ex vivo cells, whereas in vitro cells had higher frequency of ring, dicentric, and Robertsonian translocation classifications. Ex vivo cells showed more deletion types that represent more chromatid breaks, losses, and fragments. Cancer cells proliferate defectively and undergo clonal evolution as a result of these extensive chromosomal alterations, which are associated with DNA copy number gains and losses, as well as the overexpression of tumor suppressor genes. ⁴⁵ , ⁴⁶ Deficient DNA repair has been demonstrated to contribute to carcinogenesis in many cancers, particularly breast cancers. ⁴⁷

When normalized to 100 cells, the ex vivo cells exhibited approximately 7.5% more aberrations than the in vitro cells. Although only slightly greater in number, the frequency of the different classifications of aberrations was substantially different (p < 0.0001, Fisher's exact test). Structural abnormalities can correlate with more aggressive breast cancer phenotypes clinically. ⁴⁸ Other work with a murine mammary tumor cell line showed that an increase in single strand DNA gaps increased genomic instability in DNA ligase 3 (LIG3) deficient cells, with the genomic instability promoting cancer progression. ⁴⁹ Although we demonstrate substantially different types of aberrations between in vivo and ex vivo cells, further work is required to elucidate which of these aberrations, if any, alter cancer aggressiveness. However, chromosome translocations are particularly associated with the fusion of oncogenes and activation of oncogenic proteins, ⁵⁰ and more specifically with breast cancer initiation in preclinical models. ⁵¹ In our findings, chromosomal translocations were observed in both in vitro cells and ex vivo cells but were proportionally much lower in the ex vivo cells.

In our work, tumor tissue cells were collected and cryopreserved in liquid nitrogen. For cultivation, ex vivo cell vials were swiftly thawed from liquid nitrogen and subsequently transferred into cell culture media. These procedures were carried out to ensure consistency with in vitro cell culture. Cryopreservation can have an effect on stem cell‐like phenotypes, resulting in a lower stem cell yield. ⁵² According to Perrone et al., ⁵³ CD44⁺/CD24⁻ CSC were found in 91% of tumor samples, and flow cytometry analysis revealed that their percentages in five different human breast cancer samples to be heterogeneous, being 1.64%, 0.54%, 0.44%, 0%, and 1.09%. In our study, flow cytometry analysis utilized cell suspensions from both in vitro and ex vivo cells. The study's results also demonstrated the presence of CD44⁺/CD24⁻ CSCs in both in vitro cultured cells and ex vivo cells from all three experimental mice, with proportions of 0.42% ± 0.16%, 0.28% ± 0.05%, 0.61% ± 0.04%, and 0.36% ± 0.10%, respectively (Figure 3B). The stem cell‐like populations can be rare in breast cancers of epithelial origin. Mohammadalipour et al. ⁵⁴ determined almost undetectable numbers of CD44⁺/CD24⁻ cells for two epithelial derived cell lines, whereas two mesenchymal cell lines exhibited 77% and 82% of the cell population. The low values are consistent with our data for the 4T1 cells, yet despite the low number of CSCs, the BALB/c 4T1 model of triple negative breast cancer is highly aggressive.

Breast CSCs are not static entities but represent dynamic phenotypic states regulated by cytokine signaling, metabolic adaptation, and microenvironmental cues. Huang et al. ⁵⁵ highlighted breast CSCs as major mediators of radio‐ and chemoresistance in TNBC, showing that quiescent CSCs persist after cytotoxic stress and reinitiate tumor growth.

At the systems level, Pan et al. ⁵⁶ integrated cross‐tumor data showing that metabolic reprogramming, immune crosstalk, and inflammatory signaling are universal mechanisms maintaining CSC populations. In TNBC, metabolic plasticity, particularly the switch between glycolytic and oxidative states, allows CSCs to survive nutrient deprivation and evade immune clearance. Inflammatory mediators such as IL‐6, STAT3, and NF‐κB not only enhance self‐renewal but also induce expression of immune‐evasion molecules (PD‐L1, CD47), linking CSC persistence with immune suppression. Clinically, Roosta et al. ⁵⁷ refined this paradigm by identifying a CD44⁺/CD24⁻ subset associated with poor prognosis. The frequency of CD44⁻/CD24⁻ phenotype has been shown to be a dominant factor in aggressive TNBC, correlating with tumor size and metastasis. ³⁶

Recent cytogenetic studies have identified recurrent large‐scale chromosomal arm losses in breast cancer, notably 4p deletion in basal‐like subtypes, as reported in a 2024 genome wide analysis. ⁵⁸ Similarly, loss of 16q and gain of 1q remain among the most frequent chromosomal alterations across breast cancer cohorts, reflecting a conserved pattern of unbalanced structural rearrangements observed in multiple datasets.

The formation of dicentric chromosomes has been indirectly supported through mechanistic studies elucidating the breakage–fusion–bridge (BFB) cycle, a major source of genomic instability and focal gene amplification. ⁵⁹ In this process, double‐strand breaks initiate end‐to‐end chromosome fusion, generating dicentric chromosomes that form anaphase bridges during cell division.

The 4T1 cell line is widely employed as a preclinical model of triple‐negative breast cancer (TNBC) because it lacks expression of estrogen receptor (ER), progesterone receptor (PR), and HER2, and exhibits a highly metastatic phenotype. Nevertheless, it remains a murine mammary carcinoma model and therefore cannot fully reproduce the biological and molecular complexity of human TNBC. Comparative transcriptomic and genomic profiling studies have shown that although 4T1 tumors share several hallmark features with human TNBC subtypes such as epithelial–mesenchymal transition (EMT) signatures, high proliferation indices, and immune‐suppressive microenvironments, there is considerable divergence in gene expression programs, mutational spectra, and chromosomal landscapes. ⁶⁰ For example, pathways involved in DNA damage response, immune evasion, and metabolic adaptation are often differentially regulated between murine and human tumors, reflecting species‐specific tumor evolution and microenvironmental pressures.

Moreover, fundamental differences between the murine and human immune systems impose additional translational constraints. Even in syngeneic hosts, the murine immune repertoire, myeloid cell composition, cytokine milieu, and effector mechanisms differ substantially from those in human breast cancer. The 4T1 model is characterized as poorly immunogenic, with a prominent accumulation of myeloid‐derived suppressor cells (MDSCs), macrophages, and neutrophil‐like populations that promote immunosuppression and tumor progression. Although these features mimic aspects of human TNBC immunobiology, the specific proportions, phenotypes, and activation states of these cells are not directly comparable to human immune subsets, particularly in the context of T‐cell infiltration, NK‐cell cytotoxicity, and checkpoint molecule expression. ⁶⁰

Finally, the implantation site exerts a profound influence on tumor progression and metastatic behavior. Subcutaneous (flank) 4T1 models, while technically convenient and allowing serial tumor volume monitoring, often yield fewer metastases or exhibit altered organ tropisms compared with orthotopic (mammary fat pad) implantation. ⁶¹ Orthotopic models more faithfully reproduce spontaneous metastatic dissemination to clinically relevant sites such as the lung, liver, bone, and brain. In contrast, flank tumors may display reduced vascularization, distinct stromal composition, and a different immune contexture, thereby limiting the interpretation of cancer stem cell (CSC) frequency, epithelial‐mesenchymal plasticity, and metastatic correlation.

5. CONCLUSION

Murine 4T1 cancer cells share various characteristics with TNBC. This study has shown preservation of rare stem cell‐like phenotypes during the growth of 4T1 tumors in vivo. It has also shown an evolution of chromosomal aberrations in terms of structure and number once the tumor has developed in vivo. The 4T1, BALB/c model is extensively used as a model of triple negative breast cancer. Our data add to the thorough characterization of this model to support pre‐clinical developments.

AUTHOR CONTRIBUTIONS

Quy Van‐Chanh Le: Data curation; formal analysis; investigation; visualization; writing – original draft; writing – review and editing. Zsuzsa S. Kocsis: Conceptualization; data curation; formal analysis; methodology; writing – original draft; writing – review and editing. Sisi Liang: Data curation. Yanrui Li: Data curation. Briony L. Gliddon: Data curation; methodology; supervision; writing – review and editing. Sheree Bailey: Formal analysis; methodology; supervision; writing – review and editing. Anh Thai‐Quynh Nguyen: Data curation; investigation. Tyron Turnbull: Project administration; supervision. Stuart M. Pitson: Resources; supervision; writing – review and editing. Zsolt Jurányi: Methodology; resources; supervision; writing – review and editing. Hien Le: Conceptualization; supervision; writing – review and editing. Ivan Kempson: Conceptualization; data curation; formal analysis; funding acquisition; methodology; project administration; resources; supervision; writing – original draft; writing – review and editing.

FUNDING INFORMATION

This research was supported by Tour de Cure, Pioneering Grant RSP‐253‐18/19. Z.S.K. was supported by the National Research, Development and Innovation Fund of the Ministry of Culture and Innovation under the National Laboratories Program (National Tumor Biology Laboratory (2022‐2.1.1‐NL‐2022‐00010)); the Hungarian Thematic Excellence Program (TKP2021‐EGA‐44). S.M.P. was supported by a Senior Research Fellowship (1156693) from the National Health and Medical Research Council of Australia.

CONFLICT OF INTEREST STATEMENT

The authors declare no conflict of interest.

ETHICS STATEMENT

All work conducted here was approved by the University of South Australia Animal Ethics Committee (U49‐19 and U34‐21).

Supporting information

Figure S1. Representative metaphase spreads highlighting various chromosomal aberrations.

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.