Robust and Cost-Effective Pharmacological Immunosuppression Enables Establishment of Nigerian Patient-Derived Xenograft (PDX) Models of Triple-Negative Breast Cancer (TNBC) in Low-Resource Settings

Robust and Cost-Effective Pharmacological Immunosuppression Enables Establishment of Nigerian Patient-Derived Xenograft (PDX) Models of Triple-Negative Breast Cancer (TNBC) in Low-Resource Settings | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Robust and Cost-Effective Pharmacological Immunosuppression Enables Establishment of Nigerian Patient-Derived Xenograft (PDX) Models of Triple-Negative Breast Cancer (TNBC) in Low-Resource Settings Uzoamaka Okoli, Michael Okafor, Frank Achi, Nkoyo Nubila, Callistus Iheme, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6913154/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Genetically immunodeficient mouse models such as NOD/SCID and nude strains are widely used in cancer research for establishing patient-derived xenografts (PDXs). However, these models are expensive, require specialized facilities, and do not adequately reflect human immune-tumour interactions. This study aimed to develop a cost-effective and immunologically relevant pharmacological immunosuppression protocol in immunocompetent mice for the engraftment of triple-negative breast cancer (TNBC) xenografts in low-resource settings. Methods Female albino mice were treated with either cyclosporin A (35 mg/kg) plus ketoconazole (10 mg/kg) daily for five days, or cyclophosphamide (100 mg/kg) on alternate days (Days 0, 2, and 4). Haematological parameters, body weight, spleen weight, and feeding behaviour were monitored to assess immunosuppression and toxicity. A combined staged protocol was subsequently developed: cyclosporin A + ketoconazole for 5 days followed by cyclophosphamide (200 mg/kg on Day 6 and 100 mg/kg on Day 8), with orthotopic TNBC tissue implantation on Day 9. Results Cyclosporin A + ketoconazole significantly reduced total white blood cell, and lymphocyte counts by 65% and 75%, respectively (P < 0.05 and P < 0.01). Cyclophosphamide alone induced a 50% lymphocyte reduction with minimal toxicity. The combined protocol enabled a 30% tumour take rate at first passage (P0) and 80% at second passage (P1). Recapitulates equal expression of Molecular analysis confirmed over 80% similarity between xenografts and the primary tumour. Conclusion This is the first article which demonstrates breast cancer patient derived xenograft in Nigeria and a pharmacologically induced immunosuppression regimen can support effective TNBC xenograft establishment in immunocompetent mice. The protocol is robust, cost-effective, and more accessible than genetically modified models, making it well-suited for use in resource-limited research settings. Biological sciences/Biochemistry Biological sciences/Biological techniques Biological sciences/Cancer Biological sciences/Molecular biology Health sciences/Diseases Health sciences/Medical research Health sciences/Oncology Figures Figure 1 Figure 2 Figure 3 INTRODUCTION The engraftment of patient-derived tumours into immunodeficient mice (also called nude mice), particularly nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, which exhibit both nude and severe combined immunodeficiency phenotypes, is a well-established model widely employed in cancer research for evaluating tumour growth, therapeutic response, and molecular profiling[ 1 – 3 ]. However, while these models support xenografts, they present limitations, especially in replicating the complex human immune landscape. Genetically immunodeficient mice lack key immune cell populations and thus fail to reflect the nuanced interactions between tumours and a partially functional immune system, which are often relevant in human patients. Although humanized mouse models have been developed to partially overcome this limitation by reconstituting human immune components, these models are costly, technically demanding, and require specialized facilities. These factors limit their feasibility, particularly in low-resource settings[ 4 – 6 ]. As such, there remains a critical gap in the availability of affordable, immunologically relevant in vivo models for cancer research in resource-limited environments. Pharmacological immunosuppression of immunocompetent mice presents a viable alternative to genetically immunodeficient and humanized models. This approach offers several advantages, especially in low-resource settings where access to specialized animal strains and infrastructure is limited. Immunocompetent mice subjected to pharmacological immunosuppression are robust, readily available, cost-effective, and do not require specialized transportation or housing facilities. In this study, immunosuppression was induced through sequential monotherapy or combination treatments using agents such as cyclophosphamide (CyX), cyclosporine A (CyA), and ketoconazole. CyX is a DNA-alkylating agent that impairs lymphocyte proliferation by interfering with DNA replication. Notably, T-suppressor cells are somewhat sensitive, whereas T-helper cells remain relatively resistant to the immunosuppressive effects of CyX. CyA, a calcineurin inhibitor, disrupts T-cell signalling by preventing the transcription of key cytokine genes, such as IL-2 and IL-4, thereby inhibiting T-cell activation and effector function[ 7 , 8 ]. In addition to impairing T-cell responses, CyA has been shown to deplete B lymphocytes across various species, including mice, guinea pigs, and humans [ 9 ]. CyA is metabolized in the liver via the cytochrome P450 enzyme system. The antifungal agent ketoconazole, a known CYP450 inhibitor, can significantly reduce CyA clearance when co-administered [ 10 , 11 ]. This pharmacokinetic interaction enhances and prolongs CyA-mediated immunosuppression, allowing for more efficient immune suppression at lower CyA doses [ 12 , 13 ]. Compared with nude mice, pharmacologically immunosuppressed models better preserve the physiological immune context and enable the study of cancer progression and treatment in an immunomodulated but otherwise intact host. Immunosuppression is considered successful when leukopenia, depletion of T and B cells, and a reduction in lymphoid organ mass are observed. Importantly, such a model could increase accessibility to preclinical cancer testing platforms in under resourced regions and offer a more physiologically relevant context to study tumour immune dynamics in patients with functional, although compromised, immunity. We hypothesize that pharmacologically induced immunosuppression in immunocompetent mice will sufficiently mimic immunodeficient conditions to permit successful tumour engraftment while preserving essential elements of immune function. In this study, we aimed to develop a robust and reproducible pharmacological protocol for inducing immunosuppression in mice to facilitate the engraftment of patient-derived breast tumour xenografts. Specifically, our objectives are to establish a safe and effective immunosuppressive regimen using CyX, CyA, and ketoconazole; to quantify the degree of immunosuppression via established haematological and immunological parameters; and to validate tumour engraftment efficiency in treated mice. All animal procedures were performed in accordance with institutional and international guidelines for the care and use of laboratory animals. METHODS Ethical clearance This study was approved by the College of Medicine Research Ethics Committee, University of Nigeria (Approval No. 065/03/2019). All procedures involving animals adhered to institutional and international guidelines for the care and use of laboratory animals. Animals Male and female albino mice were obtained from the Animal House, Department of Pharmacology and Therapeutics, College of Medicine, University of Nigeria, Nsukka. The animals were internally bred and maintained under standardized conditions to ensure a consistent genetic background. Healthy nulliparous female albino mice, aged 4 to 6 weeks and weighing 18–22 grams, were selected for this study. All animals were housed in polypropylene cages with stainless-steel wire lids, bedded with clean wood shavings, and maintained under controlled environmental conditions (22 ± 2°C, 50–60% relative humidity, and a 12-hour light/dark cycle) and further care measures were implemented (Supplementary Fig. 1). The mice were provided autoclaved standard rodent chow and water ad libitum. All procedures involving animals were carried out in accordance with institutional and international guidelines for the care and use of laboratory animals. Chemicals and reagents Cyclosporin A, ketoconazole, and cyclophosphamide, were purchased from Tocris, Bio-Techne (Minneapolis, USA). Ciprofloxacin was purchased from a local pharmacy. All other chemicals and reagents used were of analytical grade and procured from standard commercial sources. Determination of the Safe Level of Induced Immunosuppression in Mice To determine the safe level of immunosuppression induced by cyclophosphamide, cyclosporin A, and ketoconazole, a significantly modified protocol based on the methods described by Hou et al.[ 14 ] and Jivrajani et al.[ 15 ] was employed. Female albino mice aged 4–6 weeks were randomly divided into four groups (n = 15 per group). All the mice were prophylactically treated with 17 mg/kg ciprofloxacin. Group 1 (CYA + Keto Treated):- This group received cyclosporin A at a dose of 35 mg/kg via intraperitoneal (i.p.) injection and ketoconazole at 10 mg/kg by oral gavage daily for 14 consecutive days. Group 2 (CyX-treated): -This group received cyclophosphamide at a dose of 100 mg/kg subcutaneously, which was administered three times at two-day intervals (days 0, 2, and 4). Group 3 (Vehicle Control for Group 1): -This group received a combination of 10% DMSO and 90% olive oil via oral gavage for 14 days. Group 4 (Vehicle Control for Group 2): -This group received distilled water subcutaneously at the same time points as the cyclophosphamide-treated group. All animals were housed under standard conditions and provided autoclaved food pellets and water ad libitum throughout the study. Clinical signs of toxicity Throughout the treatment period, the mice were monitored daily for clinical signs of toxicity or distress, including changes in body weight, fur texture, behaviour, and feeding patterns observed at 30 min, 4 h, 24 h, 48 h, 7 days and 14 days. Haematological analysis Peripheral blood was collected via cardiac puncture from mice treated with cyclosporin A and ketoconazole, along with the corresponding vehicle control group, on Days 5, 10, and 15 of treatment. For the cyclophosphamide-treated group and its vehicle control, blood was collected on Days 2, 4, and 6, corresponding to the dosing schedule. The collected blood samples were immediately analysed via an automated haematology analyser (Dymind DH36, Guangzhou, China). The following haematological parameters were assessed: total white blood cell (WBC) count; lymphocyte count; neutrophil count; red blood cell (RBC) count; haematocrit (HCT); haemoglobin (Hb) concentration; and platelet count. Body weight monitoring Body weight was recorded prior to the initiation of treatment (day 0), at two-day intervals during the treatment period, and on the final day following treatment completion. This was carried out to monitor general health status, assess potential toxicity, and evaluate treatment-related physiological changes. A body weight loss of ≥ 15% from baseline was predefined as a humane endpoint, prompting immediate review and, if necessary, early euthanasia in accordance with institutional animal welfare guidelines. Feeding Pattern The estimated food consumption (EFC) was determined by measuring the difference between the initial quantity of food provided (Fi) and the amount of food remaining (Fr) after 24 hours. Food intake was assessed daily throughout the treatment period via the following formula: EFC = Fi − Fr Spleen collection and relative spleen weight calculation On day 15, the mice were euthanized by cervical dislocation, and the spleens were harvested immediately postmortem. Each spleen was weighed immediately after dissection to obtain the spleen weight (SW), and the final body weight (FBW) of each animal was recorded. The relative spleen weight was calculated as a percentage of the total spleen weight to the total body weight via the following formula: Relative Spleen Weight (%) = [SW (mg)/FBW (g)] × 100 TNBC PDX models establishment To validate the proposed pharmacological immunosuppression protocol, viable frozen-thawed breast cancer tissue fragments were coated in Cultrex® BME Type 3 to enhance tumour take rate prior to implantation[ 16 ] This frozen thawed tissue were accessed for RNA viability (Supplementary Table 2 and Supplementary Fig. 3).The fragments (approximately 2 mm³ in size) were surgically implanted into the cleared inguinal mammary fat pads of 10 pharmacologically immunosuppressed female mice, following previously described procedures [ 3 , 4 ]. Successfully engrafted tumours were subsequently passage into an additional cohort of 10 immunosuppressed mice. Tumour growth was monitored twice weekly. Once palpable, tumour dimensions were measured using a Vernier calliper, and volume was calculated using the formula: (length × width²)/2. Engraftment was defined as tumour growth reaching a volume between 70 mm³ and 150 mm³.Mice that were successfully engrafted with tumour growth size reaching up to 4000mm 3 were anaesthetised and the tumour (P 0 ) was removed for subsequent passages (P 1 ). Supplementary Fig. 4, illustrates workflow of cryopreservation of breast cancer tissue to xenograft development. After tumour removal, the mice were sacrificed by cervical dislocation and autopsy was done to determine possible tumour metastasis. Histological and Immunological analysis Primary and xenograft breast and bone tissue samples were fixed in 10% buffered formaldehyde, further processed, embedded in paraffin wax and sectioned. The sections were stained with haematoxylin and eosin as described by Fischer, 2005. The cut sections on slides were studied under a light microscope and images were further interpreted by a Consultant Histopathologist Immunohistochemistry for ER, PR, and HER2 Manual immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded (FFPE) breast tissue sections, as described by Ramos-Vara (2005). Briefly, sections were cut at 4 µm, mounted on BOND Plus adhesive slides (Leica Biosystems), and baked at 65°C for 1 hour. Slides were dewaxed in xylene and rehydrated through a graded ethanol series. Antigen retrieval was carried out using 0.01 M sodium citrate buffer (pH 6.0) in a pressure cooker at 125°C for 15 minutes under 103 kPa, followed by natural cooling. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes, and non-specific binding was blocked using 5% bovine serum albumin (BSA) for 30 minutes. Primary antibodies used were Oestrogen Receptor (ER), Clone 6F11 (Cat. No. NCL-L-ER-6F11); Progesterone Receptor (PR), Clone 312 (Cat. No. NCL-L-PGR-312); and HER2, Clone CB11 (Cat. No. NCL-L-CB11), all from Leica Biosystems. Slides were incubated overnight at 4°C with primary antibodies, then treated with a horseradish peroxidase-conjugated secondary antibody using the Novolink™ Polymer Detection System (Cat. No. RE7290-CE) for 30 minutes. Staining was visualized with DAB chromogen, followed by haematoxylin counterstaining and differentiation in 1% acid alcohol. Slides were dehydrated, cleared in xylene, and mounted with neutral resin. ER and PR expression were scored using the Allred scoring system, while HER2 was evaluated based on ASCO/CAP 2018 guidelines. All slides were independently reviewed by a certified consultant pathologist Real-Time Quantitative PCR (RT-qPCR) Analysis RT-qPCR was performed to evaluate the genomic correlation between primary breast tumours and corresponding tumour xenografts. Seven breast cancer-associated genes were selected based on their reported involvement across tumour grades (Gabrovska et al., 2012): RPL13A, CXCL16, CLDN10, CDC42EP3, EPSTI1, PALMD, and ZAN. GAPDH housekeeping gene, was used as endogenous control for normalization. (Supplementary Table 1 for primers) Total RNA was extracted from mechanically homogenized tumour tissues using guanidine isothiocyanate lysis, and cDNA was synthesized by random hexamer priming. Primer sequences for both target and reference genes were adopted from Gabrovska et al. (2012) and validated using the UCSC Genome Browser ( http://genome.ucsc.edu/ ). Primer details are provided in Supplementary Table X. RT-qPCR was conducted using TaqMan chemistry (Applied Biosystems) on the Rotor-Gene 6000 system (Corbett Research/Qiagen). Each 20 µL reaction contained 300 nM of each primer, 120 ng of cDNA, and 2× SYBR Green Master Mix (Bio-Rad). Cycling conditions included an initial denaturation at 95°C for 10 minutes, followed by 45 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. A final melt curve was run from 72–95°C, increasing by 0.2°C per step. All reactions were performed in quadruplicate. Relative gene expression was analysed using the 2^−ΔΔCt method (Livak and Schmittgen, 2001). Results were compared between xenograft and primary tumour samples to assess consistency of gene expression profiles across models. RESULTS Pharmacological Immunosuppression induction WBC counts declined progressively in the CYA+Keto group, reaching a nadir on Day 10 with partial recovery by Day 15. However, two-way ANOVA revealed no statistically significant differences ( P > 0.05) between groups (Figure 1a). A marked reduction in lymphocyte count was observed by Day 5, with the lowest levels recorded on Day 10. Two-way ANOVA revealed a significant treatment effect (P = 0.0027), confirming a statistically significant immunosuppressive response to CYA+Keto. (Figure 1b). Although a transient decrease in the WBC count was observed by Day 4, two-way ANOVA indicated no significant treatment effect (P = 0.3009), suggesting limited suppression of total WBCs (Figure Ic). Cyclophosphamide induced a significant decrease in the lymphocyte count by Day 4, followed by partial recovery on Day 6. Two-way ANOVA revealed significant effects of treatment ( P = 0.0108), time ( P = 0.0116), and their interaction ( P = 0.0450), indicating a time-dependent immunosuppressive effect (Figure 1d). Physiological effect of induced immunosuppression Feed consumption per mouse declined progressively over the 14-day treatment period with cyclosporin A and ketoconazole. Linear regression analysis revealed a significant downwards trend (slope = –0.0607, R² = 0.8910), indicating treatment-associated suppression of appetite or metabolic alteration. (Figure 2a). In contrast to the CYA+Keto group, CyX-treated mice presented only a slight, nonsignificant decrease in feed consumption over the 6-day observation window (slope = –0.0125, R² = 0.3827), suggesting minimal acute impact on feeding behaviour. (Figure 2b). Body weight was significantly lower in the CYA+Keto-treated group than in the vehicle control group (mean difference = –6.46 g, P = 0.0121), with the most pronounced weight loss observed around Day 15. The treatment effect accounted for 56.6% of the variance in body weight (R² = 0.5655) (Figure 2c). Compared with control mice, cyclophosphamide-treated mice presented a moderate but statistically significant reduction in body weight (mean difference = –2.64 g, P = 0.0044, R² = 0.6580), with partial recovery by Day 8. The variance in weight was not significantly different between the groups. (Figure 2d). Although a visual trend toward reduced spleen weight was observed in both the CyX- and CYA+Keto-treated groups compared with the baseline group, repeated-measures ANOVA revealed no statistically significant differences (P = 0.1494), (Figure 2e). Induced Immunosuppression validation via TNBC PDX establishment We established a patient-derived xenograft (PDX) from a Nigerian female patient following neoadjuvant chemotherapy. Tumour tissues were subcutaneously implanted into NSG mice. The first passage (P0) yielded a 30% tumour take rate (3/10), and the xenografts exhibited progressive tumour growth over 75 days, reaching an average volume of ~4700 mm³ (Fig. 3A). Second passage (P1) showed improved uptake of 80% (8/10), with synchronized tumour outgrowth among biological replicates (Fig. 3B). Histological comparison using haematoxylin and eosin (H&E) staining confirmed architectural similarities between the primary tumour and the xenografted tissue, with preserved breast lobules and stromal organization (Fig. 3Ci &ii). Immunohistochemical (IHC) analysis validated the triple-negative phenotype of the xenografts, with negative staining for oestrogen receptor (ER), progesterone receptor (PR), and HER2, consistent with the original tumour profile (Fig. 3Ciii). Remarkably, two mice in the P0 cohort (TNPDX0c and TNPDX0d) developed spontaneous metastasis. TNPDX0c death was recorded on the 79 th day of xenograft (Supplementary figure 2) Dissection of TNPDX0d and histological analysis revealed tumour colonization within the bone, accompanied by osteoclast activation (Fig. 3E), reflecting aggressive tumour behaviour. To further assess the fidelity of the PDX model, quantitative PCR was performed on the primary tumour and matched P0 xenografts. Expression patterns of RPL13A, CXCL16, CLDN10, CDC42EP3, EPSTI1, PALMD, ZAN and GAPDH genes were conserved between the primary and xenograft tissues, confirming transcriptional stability (Fig. 3E). Statistical analysis The data are presented as the means ± SEMs (or SDs), and statistical comparisons were performed via one-way ANOVA, unpaired t tests and linear regression. The graph shows the means and SEMs of 5 independent determinations, and the data were analysed for significant differences from the control via one-way ANOVA (GraphPad Prism 10). P > 0.05 DISCUSSION While nude mice remain widely used for xenograft establishment, they present several limitations, including the absence of functional immune cells, special housing requirements, transportation difficulties, and increased costs. In contrast, pharmacologically immunosuppressed mice are robust, readily available, and more affordable. Importantly, they retain partial immune functionality, which more closely mimics the tumour–immune interactions seen in human cancers. This model presents a cost-effective, immunologically relevant, and technically accessible alternative to genetically immunodeficient systems. By retaining partial immune functionality, pharmacologically immunosuppressed mice may offer a more physiologically reflective environment for studying tumour immune interactions an essential consideration for preclinical drug testing, especially in immune-modulatory and immunotherapy research. Moreover, its use in female mice, in contrast to the predominantly male-focused literature, broadens its relevance to breast cancer research [17–20]. To optimize xenograft take-up, we adapted and modified the immunosuppression protocol described by Jivrajani et al. [15], which had primarily been applied in male mice using non-breast cancer tumours. While previous protocols achieved high take rates (up to 100%) using various combinations of cyclosporin A (CsA), ketoconazole, cyclophosphamide (CyX), and procarbazine in male mice, such strategies were rarely validated in female mice or with breast tumours. Given the known challenges of establishing PDXs from breast cancers in females, we developed a staged immunosuppressive regimen tailored for this context. Our optimized protocol involved pretreatment with 35 mg/kg CsA and 10 mg/kg ketoconazole for 5 days, followed by cyclophosphamide on Days 6 and 8. This combination led to significant reductions in total WBC and lymphocyte counts (65% and 75%, respectively) by Day 5, confirming effective but reversible immunosuppression. Although CsA + ketoconazole treatment caused mild weight loss and physiological stress, it remained tolerable. In contrast, cyclophosphamide-only treatment was associated with less toxicity and consistent suppression of lymphocytes with better tolerability and a lower impact on body weight and feeding behaviour (Table 1; Supplementary Table 2). Using this pharmacological immunosuppression protocol, we achieved a 30% tumour take rate at first passage (P0) and 80% in second passage (P1) (Fig. 3A–B). These results are consistent with published data indicating higher engraftment success for TNBC compared to other breast cancer subtypes [21–23]. Importantly, this represents the firstreport of a TNBCPDX model established from a Nigerian patient using pharmacologically immunosuppressed female mice. Histologically, the xenograft tumours closely recapitulated the patient’s tumour in glandular morphology and stromal composition, as confirmed by H&E staining (Fig. 3Ci–ii). Immunohistochemistry showed that ER, PR, and HER2 remained negative in both primary and xenograft tumours (Fig. 3Ciii), validating maintenance of the triple-negative phenotype. Molecular fidelity was further assessed by qPCR analysis of seven breast cancer–associated genes (RPL13A, CXCL16, CLDN10, CDC42EP3, EPSTI1, PALMD, ZAN). Expression profiles between the primary tumour and xenografts showed minimal variation (fold changes between 0.01 and 1.2), indicating that key transcriptional programs were preserved despite xenotransplantation (Fig. 3E). Notably, CXCL16 and EPSTI1, which are implicated in TNBC invasiveness and immune evasion, maintained stable expression, suggesting that the immunosuppressed microenvironment did not disrupt crucial tumour signalling pathways [24–26]. Strikingly, spontaneous bone metastases were observed in two P0 mice. Gross and histological evaluation revealed tumour infiltration of bone and osteoclastic activity (Fig. 3D), underscoring the aggressive nature of the TNBC model and its utility for studying metastatic progression in vivo an uncommon but highly valuable feature in PDX studies. Limitations While our findings are promising, several limitations must be acknowledged. The xenografts were derived from one patient, limiting generalizability. Future work will involve establishing a diverse panel of PDX models from multiple Nigerian patients to reflect inter-tumoral heterogeneity. Although we assessed haematological indices and spleen size, deeper immunophenotyping (e.g., T/B/NK cell subsets via flow cytometry) was not performed. This would strengthen the mechanistic understanding of immune modulation. The study focused on model establishment. Follow-up studies will assess drug responses and survival outcomes, which are critical for translational validation. While spontaneous bone metastases were observed, we did not evaluate molecular mechanisms of dissemination or quantify metastasis frequency. Incorporating bioluminescence imaging could address this. The protocol induced transient immune suppression suitable for tumour engraftment, but the long-term immune consequences were not assessed. Chronic immunomodulation studies would clarify safety and model stability. Despite these limitations, our study provides a foundational platform for accessible and immunologically relevant TNBC modelling in low-resource contexts. In conclusion, this study provides a validated scalable and cost-effective alternative to genetically immunodeficient models for TNBC PDX development. Our pharmacologically immunosuppressed female mouse model successfully supports engraftment of Nigerian patient-derived TNBC, preserves tumour architecture and molecular features, and allows for metastasis modelling. This platform is particularly suitable for under-resourced research environments and offers a valuable tool for studying racial disparities, immune evasion, and therapeutic resistance in aggressive breast cancer. Abbreviations ALS – Antilymphocyte Serum ANOVA – Analysis of Variance BMC – Bone Marrow Cell CYP450 – Cytochrome P450 CyA – Cyclosporin A CyX – Cyclophosphamide DMSO – Dimethyl Sulfoxide EFC – Estimated Food Consumption ER+ – Oestrogen Receptor Positive FBW – Final Body Weight Fi – Initial Quantity of Food Fr – Remaining Quantity of Food HCT – Haematocrit Hb – Haemoglobin IL-2 / IL-4 / IL-10 – Interleukin 2 / 4 / 10 i.p. – Intraperitoneal Keto - Ketoconazole LNCap – Lymph Node Carcinoma of the Prostate NOD/SCID – Nonobese Diabetic / Severe Combined Immunodeficient PDX – Patient-Derived Xenograft PCH – Procarbazine P0 / P1 – Passage 0 / Passage 1 (of xenografts) RBC – Red Blood Cell SW – Spleen Weight TNBC – Triple-Negative Breast Cancer TGF-β – Transforming Growth Factor Beta WBC – White Blood Cell Declarations Ethics approval and consent to participate All animal procedures were approved by the College of Medicine Research Ethics Committee, University of Nigeria (Approval No. 065/03/2019) and were conducted in accordance with institutional and international guidelines for the care and use of laboratory animals. Consent for publication Not applicable. Availability of data and materials The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This research was partly funded by the African Research League Cycle 2017 -2019, College of Medicine, University of Nigeria, Enugu Campus. U.O . conceived and designed the study, developed all experimental methods, led the PDX model development, and wrote the original manuscript draft. M.O. contributed to the immunosuppression protocol design and PDX development. F.A . conducted histological and immunohistochemical analyses. N.N. and C.I. supported the implementation of the immunosuppression protocol. E.N. contributed to RT-PCR experiments and provided scientific mentorship. S.O ., a consultant pathologist, evaluated tumour tissue morphology and architectural integrity . 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American Journal of Pathology 176:1229–1240 Tables Table 2: Clinical Signs for Toxicity Observation Time Point Treatment Group Posture Locomotion Fur Condition Respiration Eye/Nose Discharge Feeding Behaviour Overall Appearance 30 min CYA+Keto / CyX Normal Normal Normal Normal Normal Normal Normal 4 hr CYA+Keto / CyX Normal Normal Normal Normal Normal Normal Normal 24 hr CYA+Keto / CyX Normal Normal Normal Normal Normal Normal Normal 48 hr CYA+Keto-treated Mild change Mild change Mild change Normal Normal Mild change Mild change 48 hr CyX-treated Mild change Mild change Mild change Normal Normal Mild change Mild change Day 7 CYA+Keto-treated Moderate change Moderate change Moderate change Normal Normal Moderate change Moderate change Day 7 CyX-treated Mild change Mild change Mild change Normal Normal Mild change Mild change Day 14 CYA+Keto / CyX Normal Normal Normal Normal Normal Normal Normal Table 2: Staged safe mouse pharmacological immunosuppression regimen for xenografts Time Dose Mode of Administration Day 0 17 mg/kg Ciprofloxacin Gavage Day1-5 35 mg/kg Cyclosporin A + 10 mg/kg Ketoconazole Cyclosporin A via intraperitoneal (i.p.) injection. Ketoconazole by oral gavage. Day 6 and Day 8 100 mg/kg cyclophosphamide Subcutaneous injection Day 9 Established pharmacological immunosuppressed mice ready for xenograft Additional Declarations No competing interests reported. Supplementary Files Supplementarydata.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6913154","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":474106263,"identity":"497cdc17-5fcb-40ba-a08d-6db382e3c846","order_by":0,"name":"Uzoamaka Okoli","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYHACZgaGAgk5IN0A5koQp8VAwpiBgZE0LQyJDURrMTjAfNjgg4FF+objBxsYftQwJM5sIKiFLTlxhoFE7oYziQ2MPccYEmcTtoXH+DAPSMsNoMN4GxgS5xGrJd0AqIXxL7FakoFaEkBamEG2EHSY5GG2ZEOgXwxnAv1yWOaYhDFB7/Mdbz4s8aGiTp7v+OGDD9/U2MjOOEDIGmYk9gHiInIUjIJRMApGAUEAAO+FOpm2pUfZAAAAAElFTkSuQmCC","orcid":"","institution":"University College London","correspondingAuthor":true,"prefix":"","firstName":"Uzoamaka","middleName":"","lastName":"Okoli","suffix":""},{"id":474106264,"identity":"19197ac8-50b9-4703-8df3-0d9bfe97cdd5","order_by":1,"name":"Michael Okafor","email":"","orcid":"","institution":"University of Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Michael","middleName":"","lastName":"Okafor","suffix":""},{"id":474106265,"identity":"99714b5f-1465-44c6-919f-67dc2f5669f7","order_by":2,"name":"Frank Achi","email":"","orcid":"","institution":"University of Nigeria Teaching Hospital","correspondingAuthor":false,"prefix":"","firstName":"Frank","middleName":"","lastName":"Achi","suffix":""},{"id":474106266,"identity":"999b88c3-0080-411e-9f17-ce0133c26308","order_by":3,"name":"Nkoyo Nubila","email":"","orcid":"","institution":"University of Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Nkoyo","middleName":"","lastName":"Nubila","suffix":""},{"id":474106267,"identity":"cb91a112-d961-427b-aeab-28a542435221","order_by":4,"name":"Callistus Iheme","email":"","orcid":"","institution":"Federal University of Technology","correspondingAuthor":false,"prefix":"","firstName":"Callistus","middleName":"","lastName":"Iheme","suffix":""},{"id":474106268,"identity":"e29191be-af85-4f87-97f1-82c578a95212","order_by":5,"name":"Emmanuel Nna","email":"","orcid":"","institution":"Molecular Pathology Institute, Rangers Avenue, Independence Layout, Enugu, Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Emmanuel","middleName":"","lastName":"Nna","suffix":""},{"id":474106269,"identity":"fd37a50d-2126-44f7-a19e-babb8431b8e2","order_by":6,"name":"Samuel Ohayi","email":"","orcid":"","institution":"Enugu State University Teaching Hospital","correspondingAuthor":false,"prefix":"","firstName":"Samuel","middleName":"","lastName":"Ohayi","suffix":""},{"id":474106270,"identity":"bb80a979-2374-4357-a5c2-070dabb83e69","order_by":7,"name":"Kenneth Agu","email":"","orcid":"","institution":"University of Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Kenneth","middleName":"","lastName":"Agu","suffix":""},{"id":474106271,"identity":"c47346ee-7f50-494e-8fbc-ad884260753c","order_by":8,"name":"Iroka Udeinya","email":"","orcid":"","institution":"University of Nigeria","correspondingAuthor":false,"prefix":"","firstName":"Iroka","middleName":"","lastName":"Udeinya","suffix":""}],"badges":[],"createdAt":"2025-06-17 10:08:27","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6913154/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6913154/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":85305583,"identity":"1de7c81b-3d83-43a5-8fdf-951abd5e9670","added_by":"auto","created_at":"2025-06-24 12:39:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":57234,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffects of a combination of Cyclosporin A \u0026amp; Ketoconazole, and Cyclophosphamide on Peripheral White Blood Cell and Lymphocyte Counts in Mice. (a)\u003c/strong\u003eWhite blood cell (WBC) counts in mice treated with 35 mg/kg cyclosporin A + 10 mg/kg ketoconazole (CYA+Keto) compared with vehicle control (10% DMSO + 90% olive oil) over a 15-day treatment period.. \u003cstrong\u003e(b)\u003c/strong\u003eLymphocyte counts in the same CYA+Keto-treated mice versus vehicle control mice. \u003cstrong\u003e(c)\u003c/strong\u003e WBC counts in mice treated with 100 mg/kg cyclophosphamide (CyX) compared with those in water-treated vehicle control mice over a 6-day period. \u003cstrong\u003e(d)\u003c/strong\u003elymphocyte counts in CyX-treated mice versus control mice.. All values are expressed as the mean ± SEM (n = 3 per group). Statistical analysis was performed via two-way ANOVA; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6913154/v1/df66016523705f397cd41053.png"},{"id":85306072,"identity":"353444a0-ce15-4b8d-92ab-43131b03306f","added_by":"auto","created_at":"2025-06-24 12:47:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":65068,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhysiological Effects of Cyclosporin A + Ketoconazole and Cyclophosphamide on Feed Intake, Body Weight, and Spleen Size in Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(a)\u003cstrong\u003e \u003c/strong\u003eEffects of CYA+Keto treatment on feed consumption over time. (b) Effect of CyX treatment on feed consumption over time. (c) Effects of CYA+Keto - treatment on body weight. (d) Effects of CyX treatment on body weight. (e) Relative spleen weight in mice at baseline and following immunosuppressive treatment indicating treatment-induced splenic atrophy. These findings suggest a trend toward all the data being expressed as the means ± SEMs. Statistical significance was determined via linear regression, unpaired t tests, and repeated measures ANOVA where appropriate; \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6913154/v1/1dd796530072e57fb3fda264.png"},{"id":85305586,"identity":"7d1dc818-9dca-46cd-a342-edb74dae2362","added_by":"auto","created_at":"2025-06-24 12:39:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":796350,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEstablishment and characterization of TNBC PDX model Figure 3\u003c/strong\u003e. \u003cstrong\u003eEstablishment and Characterisation of a Nigerian Triple-Negative Breast Cancer (TNBC) Patient-Derived Xenograft (PDX) Model.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(3A)\u003c/strong\u003e First passage (P0) tumour establishment and growth progression.\u003cstrong\u003e (3Ai)\u003c/strong\u003e Tumour volume kinetics showing exponential growth after initial latency. Mean tumour volume ± SD is plotted (n=3), with a 30% engraftment rate (3/10 mice).\u003cstrong\u003e(3Aii)\u003c/strong\u003e Representative images of P0 mice (TNPDX0a, 0c, 0d) with palpable tumours. \u003cstrong\u003e(3Aiii)\u003c/strong\u003e Tumour measurement using a Vernier calliper confirms tumour burden and viability.\u003cstrong\u003e (3B)\u003c/strong\u003eSecond passage (P1) serial propagation and synchronized tumour growth.\u003cstrong\u003e (3Bi)\u003c/strong\u003etumour volume profiles of successfully engrafted P1 mice (8/10), demonstrating consistent growth across biological replicates.\u003cstrong\u003e (3Bii)\u003c/strong\u003e Representative mouse (TNPDX1i) showing external tumour, surgical resection, and excised tumour mass. \u003cstrong\u003e(3C)\u003c/strong\u003e Histopathological and molecular validation of xenograft fidelity.\u003cbr\u003e\n \u003cstrong\u003e(3Ci \u003c/strong\u003e[A-human \u0026amp; B – mouse] \u003cstrong\u003e\u0026nbsp;\u0026amp; 3Cii, \u003c/strong\u003erespectively\u003cstrong\u003e)\u003c/strong\u003e H\u0026amp;E staining showing preserved histological features, including breast lobules and stroma, between primary human TNBC and the xenograft. \u003cstrong\u003e(3Ciii)\u003c/strong\u003eImmunohistochemical analysis of ER, HER2, and PR status. Panels A, D, G = positive controls; Panels B, E, H = primary tumour (ER−, HER2−, PR−); Panels C, F, I = xenograft, confirming maintained triple-negative status. Scalebar at x40 magnification. \u003cstrong\u003e(3D)\u003c/strong\u003e Metastasis to bone in P0 xenograft-bearing mice. Gross pathology and histological section from TNPDX0c and TNPDX0d reveal bone colonization by tumour cells with osteoclast activity, indicating spontaneous metastasis and model aggressiveness. \u003cstrong\u003e(3E)\u003c/strong\u003e Gene expression comparison between primary tumour and xenograft. Quantitative PCR analysis of seven TNBC-associated genes (RPL13A, CXCL16, CLDN10, CDC42EP3, EPSTI1, PALMD, ZAN) normalized to GAPDH. Relative fold changes ranged between 0.01–1.2, indicating comparable gene expression levels between xenograft and primary tumour.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6913154/v1/9438eed53dc97ccd8a0ca998.png"},{"id":85307175,"identity":"231ece8d-a006-4c11-905c-04a2200bd090","added_by":"auto","created_at":"2025-06-24 12:55:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2191604,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6913154/v1/1006b989-65ab-4198-b28e-cbf81beb41e0.pdf"},{"id":85305585,"identity":"d58e7c41-6f39-41ce-8821-4f2ac75278cf","added_by":"auto","created_at":"2025-06-24 12:39:49","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":687678,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarydata.docx","url":"https://assets-eu.researchsquare.com/files/rs-6913154/v1/ede73af304d27177f1f5d04b.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Robust and Cost-Effective Pharmacological Immunosuppression Enables Establishment of Nigerian Patient-Derived Xenograft (PDX) Models of Triple-Negative Breast Cancer (TNBC) in Low-Resource Settings","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eThe engraftment of patient-derived tumours into immunodeficient mice (also called nude mice), particularly nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice, which exhibit both nude and severe combined immunodeficiency phenotypes, is a well-established model widely employed in cancer research for evaluating tumour growth, therapeutic response, and molecular profiling[\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, while these models support xenografts, they present limitations, especially in replicating the complex human immune landscape. Genetically immunodeficient mice lack key immune cell populations and thus fail to reflect the nuanced interactions between tumours and a partially functional immune system, which are often relevant in human patients.\u003c/p\u003e \u003cp\u003eAlthough humanized mouse models have been developed to partially overcome this limitation by reconstituting human immune components, these models are costly, technically demanding, and require specialized facilities. These factors limit their feasibility, particularly in low-resource settings[\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. As such, there remains a critical gap in the availability of affordable, immunologically relevant in vivo models for cancer research in resource-limited environments.\u003c/p\u003e \u003cp\u003ePharmacological immunosuppression of immunocompetent mice presents a viable alternative to genetically immunodeficient and humanized models. This approach offers several advantages, especially in low-resource settings where access to specialized animal strains and infrastructure is limited. Immunocompetent mice subjected to pharmacological immunosuppression are robust, readily available, cost-effective, and do not require specialized transportation or housing facilities.\u003c/p\u003e \u003cp\u003eIn this study, immunosuppression was induced through sequential monotherapy or combination treatments using agents such as cyclophosphamide (CyX), cyclosporine A (CyA), and ketoconazole. CyX is a DNA-alkylating agent that impairs lymphocyte proliferation by interfering with DNA replication. Notably, T-suppressor cells are somewhat sensitive, whereas T-helper cells remain relatively resistant to the immunosuppressive effects of CyX. CyA, a calcineurin inhibitor, disrupts T-cell signalling by preventing the transcription of key cytokine genes, such as IL-2 and IL-4, thereby inhibiting T-cell activation and effector function[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In addition to impairing T-cell responses, CyA has been shown to deplete B lymphocytes across various species, including mice, guinea pigs, and humans [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCyA is metabolized in the liver via the cytochrome P450 enzyme system. The antifungal agent ketoconazole, a known CYP450 inhibitor, can significantly reduce CyA clearance when co-administered [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. This pharmacokinetic interaction enhances and prolongs CyA-mediated immunosuppression, allowing for more efficient immune suppression at lower CyA doses [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCompared with nude mice, pharmacologically immunosuppressed models better preserve the physiological immune context and enable the study of cancer progression and treatment in an immunomodulated but otherwise intact host. Immunosuppression is considered successful when leukopenia, depletion of T and B cells, and a reduction in lymphoid organ mass are observed. Importantly, such a model could increase accessibility to preclinical cancer testing platforms in under resourced regions and offer a more physiologically relevant context to study tumour immune dynamics in patients with functional, although compromised, immunity. We hypothesize that pharmacologically induced immunosuppression in immunocompetent mice will sufficiently mimic immunodeficient conditions to permit successful tumour engraftment while preserving essential elements of immune function. In this study, we aimed to develop a robust and reproducible pharmacological protocol for inducing immunosuppression in mice to facilitate the engraftment of patient-derived breast tumour xenografts. Specifically, our objectives are to establish a safe and effective immunosuppressive regimen using CyX, CyA, and ketoconazole; to quantify the degree of immunosuppression via established haematological and immunological parameters; and to validate tumour engraftment efficiency in treated mice. All animal procedures were performed in accordance with institutional and international guidelines for the care and use of laboratory animals.\u003c/p\u003e"},{"header":"METHODS","content":"\u003cp\u003eEthical clearance\u003c/p\u003e \u003cp\u003e This study was approved by the College of Medicine Research Ethics Committee, University of Nigeria (Approval No. 065/03/2019). All procedures involving animals adhered to institutional and international guidelines for the care and use of laboratory animals.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eMale and female albino mice were obtained from the Animal House, Department of Pharmacology and Therapeutics, College of Medicine, University of Nigeria, Nsukka. The animals were internally bred and maintained under standardized conditions to ensure a consistent genetic background. Healthy nulliparous female albino mice, aged 4 to 6 weeks and weighing 18\u0026ndash;22 grams, were selected for this study.\u003c/p\u003e \u003cp\u003e All animals were housed in polypropylene cages with stainless-steel wire lids, bedded with clean wood shavings, and maintained under controlled environmental conditions (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 50\u0026ndash;60% relative humidity, and a 12-hour light/dark cycle) and further care measures were implemented (Supplementary Fig.\u0026nbsp;1). The mice were provided autoclaved standard rodent chow and water ad libitum. All procedures involving animals were carried out in accordance with institutional and international guidelines for the care and use of laboratory animals.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eChemicals and reagents\u003c/h3\u003e\n\u003cp\u003eCyclosporin A, ketoconazole, and cyclophosphamide, were purchased from Tocris, Bio-Techne (Minneapolis, USA). Ciprofloxacin was purchased from a local pharmacy. All other chemicals and reagents used were of analytical grade and procured from standard commercial sources.\u003c/p\u003e\n\u003ch3\u003eDetermination of the Safe Level of Induced Immunosuppression in Mice\u003c/h3\u003e\n\u003cp\u003eTo determine the safe level of immunosuppression induced by cyclophosphamide, cyclosporin A, and ketoconazole, a significantly modified protocol based on the methods described by Hou et al.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] and Jivrajani et al.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] was employed. Female albino mice aged 4\u0026ndash;6 weeks were randomly divided into four groups (n\u0026thinsp;=\u0026thinsp;15 per group). All the mice were prophylactically treated with 17 mg/kg ciprofloxacin. Group 1 (CYA\u0026thinsp;+\u0026thinsp;Keto Treated):- This group received cyclosporin A at a dose of 35 mg/kg via intraperitoneal (i.p.) injection and ketoconazole at 10 mg/kg by oral gavage daily for 14 consecutive days. Group 2 (CyX-treated): -This group received cyclophosphamide at a dose of 100 mg/kg subcutaneously, which was administered three times at two-day intervals (days 0, 2, and 4). Group 3 (Vehicle Control for Group 1): -This group received a combination of 10% DMSO and 90% olive oil via oral gavage for 14 days. Group 4 (Vehicle Control for Group 2): -This group received distilled water subcutaneously at the same time points as the cyclophosphamide-treated group. All animals were housed under standard conditions and provided autoclaved food pellets and water ad libitum throughout the study.\u003c/p\u003e\n\u003ch3\u003eClinical signs of toxicity\u003c/h3\u003e\n\u003cp\u003eThroughout the treatment period, the mice were monitored daily for clinical signs of toxicity or distress, including changes in body weight, fur texture, behaviour, and feeding patterns observed at 30 min, 4 h, 24 h, 48 h, 7 days and 14 days.\u003c/p\u003e\n\u003ch3\u003eHaematological analysis\u003c/h3\u003e\n\u003cp\u003ePeripheral blood was collected via cardiac puncture from mice treated with cyclosporin A and ketoconazole, along with the corresponding vehicle control group, on Days 5, 10, and 15 of treatment. For the cyclophosphamide-treated group and its vehicle control, blood was collected on Days 2, 4, and 6, corresponding to the dosing schedule.\u003c/p\u003e \u003cp\u003eThe collected blood samples were immediately analysed via an automated haematology analyser (Dymind DH36, Guangzhou, China). The following haematological parameters were assessed: total white blood cell (WBC) count; lymphocyte count; neutrophil count; red blood cell (RBC) count; haematocrit (HCT); haemoglobin (Hb) concentration; and platelet count.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eBody weight monitoring\u003c/h2\u003e \u003cp\u003eBody weight was recorded prior to the initiation of treatment (day 0), at two-day intervals during the treatment period, and on the final day following treatment completion. This was carried out to monitor general health status, assess potential toxicity, and evaluate treatment-related physiological changes. A body weight loss of \u0026ge;\u0026thinsp;15% from baseline was predefined as a humane endpoint, prompting immediate review and, if necessary, early euthanasia in accordance with institutional animal welfare guidelines.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eFeeding Pattern\u003c/h3\u003e\n\u003cp\u003eThe estimated food consumption (EFC) was determined by measuring the difference between the initial quantity of food provided (Fi) and the amount of food remaining (Fr) after 24 hours. Food intake was assessed daily throughout the treatment period via the following formula:\u003c/p\u003e \u003cp\u003eEFC\u0026thinsp;=\u0026thinsp;Fi\u0026thinsp;\u0026minus;\u0026thinsp;Fr\u003c/p\u003e\n\u003ch3\u003eSpleen collection and relative spleen weight calculation\u003c/h3\u003e\n\u003cp\u003eOn day 15, the mice were euthanized by cervical dislocation, and the spleens were harvested immediately postmortem. Each spleen was weighed immediately after dissection to obtain the spleen weight (SW), and the final body weight (FBW) of each animal was recorded. The relative spleen weight was calculated as a percentage of the total spleen weight to the total body weight via the following formula:\u003c/p\u003e \u003cp\u003eRelative Spleen Weight (%) = [SW (mg)/FBW (g)] \u0026times; 100\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eTNBC PDX models establishment\u003c/h2\u003e \u003cp\u003eTo validate the proposed pharmacological immunosuppression protocol, viable frozen-thawed breast cancer tissue fragments were coated in Cultrex\u0026reg; BME Type 3 to enhance tumour take rate prior to implantation[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] This frozen thawed tissue were accessed for RNA viability (Supplementary Table\u0026nbsp;2 and Supplementary Fig.\u0026nbsp;3).The fragments (approximately 2 mm\u0026sup3; in size) were surgically implanted into the cleared inguinal mammary fat pads of 10 pharmacologically immunosuppressed female mice, following previously described procedures [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Successfully engrafted tumours were subsequently passage into an additional cohort of 10 immunosuppressed mice.\u003c/p\u003e \u003cp\u003eTumour growth was monitored twice weekly. Once palpable, tumour dimensions were measured using a Vernier calliper, and volume was calculated using the formula:\u003c/p\u003e \u003cp\u003e(length \u0026times; width\u0026sup2;)/2. Engraftment was defined as tumour growth reaching a volume between 70 mm\u0026sup3; and 150 mm\u0026sup3;.Mice that were successfully engrafted with tumour growth size reaching up to 4000mm\u003csup\u003e3\u003c/sup\u003e were anaesthetised and the tumour (P\u003csub\u003e0\u003c/sub\u003e) was removed for subsequent passages (P\u003csub\u003e1\u003c/sub\u003e). Supplementary Fig.\u0026nbsp;4, illustrates workflow of cryopreservation of breast cancer tissue to xenograft development. After tumour removal, the mice were sacrificed by cervical dislocation and autopsy was done to determine possible tumour metastasis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eHistological and Immunological analysis\u003c/h2\u003e \u003cp\u003ePrimary and xenograft breast and bone tissue samples were fixed in 10% buffered formaldehyde, further processed, embedded in paraffin wax and sectioned. The sections were stained with haematoxylin and eosin as described by Fischer, 2005. The cut sections on slides were studied under a light microscope and images were further interpreted by a Consultant Histopathologist\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eImmunohistochemistry for ER, PR, and HER2\u003c/h2\u003e \u003cp\u003eManual immunohistochemistry (IHC) was performed on formalin-fixed, paraffin-embedded (FFPE) breast tissue sections, as described by Ramos-Vara (2005). Briefly, sections were cut at 4 \u0026micro;m, mounted on BOND Plus adhesive slides (Leica Biosystems), and baked at 65\u0026deg;C for 1 hour. Slides were dewaxed in xylene and rehydrated through a graded ethanol series. Antigen retrieval was carried out using 0.01 M sodium citrate buffer (pH 6.0) in a pressure cooker at 125\u0026deg;C for 15 minutes under 103 kPa, followed by natural cooling. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 10 minutes, and non-specific binding was blocked using 5% bovine serum albumin (BSA) for 30 minutes. Primary antibodies used were Oestrogen Receptor (ER), Clone 6F11 (Cat. No. NCL-L-ER-6F11); Progesterone Receptor (PR), Clone 312 (Cat. No. NCL-L-PGR-312); and HER2, Clone CB11 (Cat. No. NCL-L-CB11), all from Leica Biosystems. Slides were incubated overnight at 4\u0026deg;C with primary antibodies, then treated with a horseradish peroxidase-conjugated secondary antibody using the Novolink\u0026trade; Polymer Detection System (Cat. No. RE7290-CE) for 30 minutes.\u003c/p\u003e \u003cp\u003eStaining was visualized with DAB chromogen, followed by haematoxylin counterstaining and differentiation in 1% acid alcohol. Slides were dehydrated, cleared in xylene, and mounted with neutral resin.\u003c/p\u003e \u003cp\u003eER and PR expression were scored using the Allred scoring system, while HER2 was evaluated based on ASCO/CAP 2018 guidelines. All slides were independently reviewed by a certified consultant pathologist\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eReal-Time Quantitative PCR (RT-qPCR) Analysis\u003c/h2\u003e \u003cp\u003eRT-qPCR was performed to evaluate the genomic correlation between primary breast tumours and corresponding tumour xenografts. Seven breast cancer-associated genes were selected based on their reported involvement across tumour grades (Gabrovska et al., 2012): RPL13A, CXCL16, CLDN10, CDC42EP3, EPSTI1, PALMD, and ZAN. GAPDH housekeeping gene, was used as endogenous control for normalization. (Supplementary Table\u0026nbsp;1 for primers)\u003c/p\u003e \u003cp\u003eTotal RNA was extracted from mechanically homogenized tumour tissues using guanidine isothiocyanate lysis, and cDNA was synthesized by random hexamer priming. Primer sequences for both target and reference genes were adopted from Gabrovska et al. (2012) and validated using the UCSC Genome Browser (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://genome.ucsc.edu/\u003c/span\u003e\u003cspan address=\"http://genome.ucsc.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Primer details are provided in Supplementary Table X.\u003c/p\u003e \u003cp\u003eRT-qPCR was conducted using TaqMan chemistry (Applied Biosystems) on the Rotor-Gene 6000 system (Corbett Research/Qiagen). Each 20 \u0026micro;L reaction contained 300 nM of each primer, 120 ng of cDNA, and 2\u0026times; SYBR Green Master Mix (Bio-Rad). Cycling conditions included an initial denaturation at 95\u0026deg;C for 10 minutes, followed by 45 cycles of 95\u0026deg;C for 30 seconds, 60\u0026deg;C for 30 seconds, and 72\u0026deg;C for 30 seconds. A final melt curve was run from 72\u0026ndash;95\u0026deg;C, increasing by 0.2\u0026deg;C per step. All reactions were performed in quadruplicate. Relative gene expression was analysed using the 2^\u0026minus;ΔΔCt method (Livak and Schmittgen, 2001). Results were compared between xenograft and primary tumour samples to assess consistency of gene expression profiles across models.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cp\u003e\u003cstrong\u003ePharmacological Immunosuppression induction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWBC counts declined progressively in the CYA+Keto group, reaching a nadir on Day 10 with partial recovery by Day 15. However, two-way ANOVA revealed no statistically significant differences (\u003cem\u003eP\u003c/em\u003e \u0026gt; 0.05) between groups (Figure 1a). A marked reduction in lymphocyte count was observed by Day 5, with the lowest levels recorded on Day 10. Two-way ANOVA revealed a significant treatment effect (P = 0.0027), confirming a statistically significant immunosuppressive response to CYA+Keto. (Figure 1b). Although a transient decrease in the WBC count was observed by Day 4, two-way ANOVA indicated no significant treatment effect (P = 0.3009), suggesting limited suppression of total WBCs (Figure Ic). Cyclophosphamide induced a significant decrease in the lymphocyte count by Day 4, followed by partial recovery on Day 6. Two-way ANOVA revealed significant effects of treatment (\u003cem\u003eP\u003c/em\u003e = 0.0108), time (\u003cem\u003eP\u003c/em\u003e = 0.0116), and their interaction (\u003cem\u003eP\u003c/em\u003e = 0.0450), indicating a time-dependent immunosuppressive effect (Figure 1d).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePhysiological effect of induced immunosuppression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFeed consumption per mouse declined progressively over the 14-day treatment period with cyclosporin A and ketoconazole. Linear regression analysis revealed a significant downwards trend (slope = \u0026ndash;0.0607, \u003cem\u003eR\u0026sup2;\u003c/em\u003e = 0.8910), indicating treatment-associated suppression of appetite or metabolic alteration. (Figure 2a). In contrast to the CYA+Keto group, CyX-treated mice presented only a slight, nonsignificant decrease in feed consumption over the 6-day observation window (slope = \u0026ndash;0.0125, \u003cem\u003eR\u0026sup2;\u003c/em\u003e = 0.3827), suggesting minimal acute impact on feeding behaviour. (Figure 2b). Body weight was significantly lower in the CYA+Keto-treated group than in the vehicle control group (mean difference = \u0026ndash;6.46 g, P = 0.0121), with the most pronounced weight loss observed around Day 15. The treatment effect accounted for 56.6% of the variance in body weight (R\u0026sup2; = 0.5655) (Figure 2c). Compared with control mice, cyclophosphamide-treated mice presented a moderate but statistically significant reduction in body weight (mean difference = \u0026ndash;2.64 g, \u003cem\u003eP\u003c/em\u003e = 0.0044, \u003cem\u003eR\u0026sup2;\u003c/em\u003e = 0.6580), with partial recovery by Day 8. The variance in weight was not significantly different between the groups. (Figure 2d). Although a visual trend toward reduced spleen weight was observed in both the CyX- and CYA+Keto-treated groups compared with the baseline group, repeated-measures ANOVA revealed no statistically significant differences (P = 0.1494), (Figure 2e).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInduced Immunosuppression validation via TNBC PDX establishment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe established a patient-derived xenograft (PDX) from a Nigerian female patient following neoadjuvant chemotherapy. Tumour tissues were subcutaneously implanted into NSG mice. The first passage (P0) yielded a 30% tumour take rate (3/10), and the xenografts exhibited progressive tumour growth over 75 days, reaching an average volume of ~4700 mm\u0026sup3; (Fig. 3A). Second passage (P1) showed improved uptake of 80% (8/10), with synchronized tumour outgrowth among biological replicates (Fig. 3B). Histological comparison using haematoxylin and eosin (H\u0026amp;E) staining confirmed architectural similarities between the primary tumour and the xenografted tissue, with preserved breast lobules and stromal organization (Fig. 3Ci \u0026amp;ii). Immunohistochemical (IHC) analysis validated the triple-negative phenotype of the xenografts, with negative staining for oestrogen receptor (ER), progesterone receptor (PR), and HER2, consistent with the original tumour profile (Fig. 3Ciii). Remarkably, two mice in the P0 cohort (TNPDX0c and TNPDX0d) developed spontaneous metastasis. TNPDX0c death was recorded on the 79\u003csup\u003eth\u003c/sup\u003e day of xenograft (Supplementary figure 2) Dissection of TNPDX0d and histological analysis revealed tumour colonization within the bone, accompanied by osteoclast activation (Fig. 3E), reflecting aggressive tumour behaviour.\u003c/p\u003e\n\u003cp\u003eTo further assess the fidelity of the PDX model, quantitative PCR was performed on the primary tumour and matched P0 xenografts. Expression patterns of RPL13A, CXCL16, CLDN10, CDC42EP3, EPSTI1, PALMD, ZAN and GAPDH genes were conserved between the primary and xenograft tissues, confirming transcriptional stability (Fig. 3E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eanalysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data are presented as the means \u0026plusmn; SEMs (or SDs), and statistical comparisons were performed via one-way ANOVA, unpaired t tests and linear regression. The graph shows the means and SEMs of 5 independent determinations, and the data were analysed for significant differences from the control via one-way ANOVA (GraphPad Prism 10). P \u0026gt; 0.05\u0026nbsp;\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eWhile nude mice remain widely used for xenograft establishment, they present several limitations, including the absence of functional immune cells, special housing requirements, transportation difficulties, and increased costs. In contrast, pharmacologically immunosuppressed mice are robust, readily available, and more affordable. Importantly, they retain partial immune functionality, which more closely mimics the tumour–immune interactions seen in human cancers.\u003c/p\u003e\n\u003cp\u003eThis model presents a cost-effective, immunologically relevant, and technically accessible alternative to genetically immunodeficient systems. By retaining partial immune functionality, pharmacologically immunosuppressed mice may offer a more physiologically reflective environment for studying tumour immune interactions an essential consideration for preclinical drug testing, especially in immune-modulatory and immunotherapy research. Moreover, its use in female mice, in contrast to the predominantly male-focused literature, broadens its relevance to breast cancer research\u0026nbsp;[17–20].\u0026nbsp;\u0026nbsp;To optimize xenograft take-up, we adapted and modified the immunosuppression protocol described by Jivrajani et al. [15], which had primarily been applied in male mice using non-breast cancer tumours. While previous protocols achieved high take rates (up to 100%) using various combinations of cyclosporin A (CsA), ketoconazole, cyclophosphamide (CyX), and procarbazine in male mice, such strategies were rarely validated in female mice or with breast tumours. Given the known challenges of establishing PDXs from breast cancers in females, we developed a staged immunosuppressive regimen tailored for this context.\u003c/p\u003e\n\u003cp\u003eOur optimized protocol involved pretreatment with 35 mg/kg CsA and 10 mg/kg ketoconazole for 5 days, followed by cyclophosphamide on Days 6 and 8. This combination led to significant reductions in total WBC and lymphocyte counts (65% and 75%, respectively) by Day 5, confirming effective but reversible immunosuppression. Although CsA + ketoconazole treatment caused mild weight loss and physiological stress, it remained tolerable. In contrast, cyclophosphamide-only treatment was associated with less toxicity and consistent suppression of lymphocytes with better tolerability and a lower impact on body weight and feeding behaviour (Table 1; Supplementary Table 2).\u003c/p\u003e\n\u003cp\u003eUsing this pharmacological immunosuppression protocol, we achieved a 30% tumour take rate at first passage (P0) and 80% in second passage (P1) (Fig. 3A–B). These results are consistent with published data indicating higher engraftment success for TNBC compared to other breast cancer subtypes [21–23]. Importantly, this represents the firstreport of a TNBCPDX model established from a Nigerian patient using pharmacologically immunosuppressed female mice.\u003c/p\u003e\n\u003cp\u003eHistologically, the xenograft tumours closely recapitulated \u0026nbsp;the patient’s tumour in glandular morphology and stromal composition, as confirmed by H\u0026amp;E staining (Fig. 3Ci–ii). Immunohistochemistry showed that ER, PR, and HER2 remained negative in both primary and xenograft tumours (Fig. 3Ciii), validating maintenance of the triple-negative phenotype.\u003c/p\u003e\n\u003cp\u003eMolecular fidelity was further assessed by qPCR analysis of seven breast cancer–associated genes (RPL13A, CXCL16, CLDN10, CDC42EP3, EPSTI1, PALMD, ZAN). Expression profiles between the primary tumour and xenografts showed minimal variation (fold changes between 0.01 and 1.2), indicating that key transcriptional programs were preserved despite xenotransplantation (Fig. 3E). Notably, CXCL16 and EPSTI1, which are implicated in TNBC invasiveness and immune evasion, maintained stable expression, suggesting that the immunosuppressed microenvironment did not disrupt crucial tumour signalling pathways [24–26].\u003c/p\u003e\n\u003cp\u003eStrikingly, spontaneous bone metastases were observed in two P0 mice. Gross and histological evaluation revealed tumour infiltration of bone and osteoclastic activity (Fig. 3D), underscoring the aggressive nature of the TNBC model and its utility for studying metastatic progression in vivo an uncommon but highly valuable feature in PDX studies.\u003c/p\u003e\n\u003cp\u003eLimitations\u003c/p\u003e\n\u003cp\u003eWhile our findings are promising, several limitations must be acknowledged. The xenografts were derived from one patient, limiting generalizability. Future work will involve establishing a diverse panel of PDX models from multiple Nigerian patients to reflect inter-tumoral heterogeneity. Although we assessed haematological indices and spleen size, deeper immunophenotyping (e.g., T/B/NK cell subsets via flow cytometry) was not performed. This would strengthen the mechanistic understanding of immune modulation. The study focused on model establishment. Follow-up studies will assess drug responses and survival outcomes, which are critical for translational validation. While spontaneous bone metastases were observed, we did not evaluate molecular mechanisms of dissemination or quantify metastasis frequency. Incorporating bioluminescence imaging could address this. The protocol induced transient immune suppression suitable for tumour engraftment, but the long-term immune consequences were not assessed. Chronic immunomodulation studies would clarify safety and model stability. Despite these limitations, our study provides a foundational platform for accessible and immunologically relevant TNBC modelling in low-resource contexts.\u003c/p\u003e\n\u003cp\u003eIn conclusion, this study provides a validated scalable and cost-effective alternative to genetically immunodeficient models for TNBC PDX development. Our pharmacologically immunosuppressed female mouse model successfully supports engraftment of Nigerian patient-derived TNBC, preserves tumour architecture and molecular features, and allows for metastasis modelling. This platform is particularly suitable for under-resourced research environments and offers a valuable tool for studying racial disparities, immune evasion, and therapeutic resistance in aggressive breast cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eALS \u0026ndash; Antilymphocyte Serum\u003c/p\u003e\n\u003cp\u003eANOVA \u0026ndash; Analysis of Variance\u003c/p\u003e\n\u003cp\u003eBMC \u0026ndash; Bone Marrow Cell\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCYP450 \u0026ndash; Cytochrome P450\u003c/p\u003e\n\u003cp\u003eCyA \u0026nbsp;\u0026ndash; Cyclosporin A\u003c/p\u003e\n\u003cp\u003eCyX \u0026ndash; Cyclophosphamide\u003c/p\u003e\n\u003cp\u003eDMSO \u0026ndash; Dimethyl Sulfoxide\u003c/p\u003e\n\u003cp\u003eEFC \u0026ndash; Estimated Food Consumption\u003c/p\u003e\n\u003cp\u003eER+ \u0026ndash; Oestrogen Receptor Positive\u003c/p\u003e\n\u003cp\u003eFBW \u0026ndash; Final Body Weight\u003c/p\u003e\n\u003cp\u003eFi \u0026ndash; Initial Quantity of Food\u003c/p\u003e\n\u003cp\u003eFr \u0026ndash; Remaining Quantity of Food\u003c/p\u003e\n\u003cp\u003eHCT \u0026ndash; Haematocrit\u003c/p\u003e\n\u003cp\u003eHb \u0026ndash; Haemoglobin\u003c/p\u003e\n\u003cp\u003eIL-2 / IL-4 / IL-10 \u0026ndash; Interleukin 2 / 4 / 10\u003c/p\u003e\n\u003cp\u003ei.p. \u0026ndash; Intraperitoneal\u003c/p\u003e\n\u003cp\u003eKeto - Ketoconazole\u003c/p\u003e\n\u003cp\u003eLNCap \u0026ndash; Lymph Node Carcinoma of the Prostate\u003c/p\u003e\n\u003cp\u003eNOD/SCID \u0026ndash; Nonobese Diabetic / Severe Combined Immunodeficient\u003c/p\u003e\n\u003cp\u003ePDX \u0026ndash; Patient-Derived Xenograft\u003c/p\u003e\n\u003cp\u003ePCH \u0026ndash; Procarbazine\u003c/p\u003e\n\u003cp\u003eP0 / P1 \u0026ndash; Passage 0 / Passage 1 (of xenografts)\u003c/p\u003e\n\u003cp\u003eRBC \u0026ndash; Red Blood Cell\u003c/p\u003e\n\u003cp\u003eSW \u0026ndash; Spleen Weight\u003c/p\u003e\n\u003cp\u003eTNBC \u0026ndash; Triple-Negative Breast Cancer\u003c/p\u003e\n\u003cp\u003eTGF-\u0026beta; \u0026ndash; Transforming Growth Factor Beta\u003c/p\u003e\n\u003cp\u003eWBC \u0026ndash; White Blood Cell\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal procedures were approved by the College of Medicine Research Ethics Committee, University of Nigeria (Approval No. 065/03/2019) and were conducted in accordance with institutional and international guidelines for the care and use of laboratory animals.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was partly funded by the African Research League Cycle 2017 -2019, College of Medicine, University of Nigeria, Enugu Campus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eU.O\u003c/strong\u003e. conceived and designed the study, developed all experimental methods, led the PDX model development, and wrote the original manuscript draft. \u003cstrong\u003eM.O.\u003c/strong\u003e contributed to the immunosuppression protocol design and PDX development. \u003cstrong\u003eF.A\u003c/strong\u003e. conducted histological and immunohistochemical analyses. \u003cstrong\u003eN.N.\u003c/strong\u003e and \u003cstrong\u003eC.I.\u003c/strong\u003e supported the implementation of the immunosuppression protocol.\u003cstrong\u003e\u0026nbsp;E.N.\u0026nbsp;\u003c/strong\u003econtributed to RT-PCR experiments and provided scientific mentorship. \u003cstrong\u003eS.O\u003c/strong\u003e., a consultant pathologist, evaluated tumour tissue morphology and architectural integrity\u003cstrong\u003e. K.A.,\u0026nbsp;\u003c/strong\u003ea consultant surgeon, performed surgical resection of the primary tumour, facilitated patient consent, and provided clinical mentorship. \u003cstrong\u003eI.U.\u003c/strong\u003e served as the lead academic supervisor for the project. All authors reviewed the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the staff of the Animal House Facility at the Department of Pharmacology and Therapeutics, University of Nigeria, for their support in animal care and husbandry.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eLiu Y, Wu W, Cai C, Zhang H, Shen H, Han Y (2023) Patient-derived xenograft models in cancer therapy: technologies and applications. Signal Transduction and Targeted Therapy 2023 8:1 8:1\u0026ndash;24\u003c/li\u003e\n\u003cli\u003eGoto T (2020) Patient-derived tumor xenograft models: Toward the establishment of precision cancer medicine. J Pers Med 10:1\u0026ndash;14\u003c/li\u003e\n\u003cli\u003eYoshida GJ (2020) Applications of patient-derived tumor xenograft models and tumor organoids. Journal of Hematology \u0026amp; Oncology 2020 13:1 13:1\u0026ndash;16\u003c/li\u003e\n\u003cli\u003eAkkina R (2013) Human immune responses and potential for vaccine assessment in humanized mice. Curr Opin Immunol 25:403\u0026ndash;409\u003c/li\u003e\n\u003cli\u003eBrehm MA, Shultz LD, Luban J, Greiner DL (2013) Overcoming current limitations in humanized mouse research. J Infect Dis. https://doi.org/10.1093/INFDIS/JIT319,\u003c/li\u003e\n\u003cli\u003eChuprin J, Buettner H, Seedhom MO, Greiner DL, Keck JG, Ishikawa F, Shultz LD, Brehm MA (2023) Humanized mouse models for immuno-oncology research. Nature Reviews Clinical Oncology 2023 20:3 20:192\u0026ndash;206\u003c/li\u003e\n\u003cli\u003eTsuda K, Yamanaka K, Kitagawa H, Akeda T, Naka M, Niwa K, Nakanishi T, Kakeda M, Gabazza EC, Mizutani H (2012) Calcineurin Inhibitors Suppress Cytokine Production from Memory T Cells and Differentiation of Na\u0026iuml;ve T Cells into Cytokine-Producing Mature T Cells. PLoS One 7:e31465\u003c/li\u003e\n\u003cli\u003eHilchey SP, Palshikar MG, Emo JA, Li D, Garigen J, Wang J, Mendelson ES, Cipolla V, Thakar J, Zand MS (2020) Cyclosporine a directly affects human and mouse b cell migration in vitro by disrupting a hIF-1 \u0026alpha;dependent, o2 sensing, molecular switch. BMC Immunol 21:1\u0026ndash;18\u003c/li\u003e\n\u003cli\u003eGraalmann T, Borst K, Manchanda H, et al (2021) B cell depletion impairs vaccination-induced CD8 + T cell responses in a type i interferon-dependent manner. Ann Rheum Dis 80:1537\u0026ndash;1544\u003c/li\u003e\n\u003cli\u003eWeiss J, Foerster KI, Weber M, Burhenne J, Mikus G, Lehr T, Haefeli WE (2022) Does the circulating ketoconazole metabolite N-deacetyl ketoconazole contribute to the drug-drug interaction potential of the parent compound? European Journal of Pharmaceutical Sciences 169:106076\u003c/li\u003e\n\u003cli\u003eGilani B, Cassagnol M (2023) Biochemistry, Cytochrome P450. StatPearls \u003c/li\u003e\n\u003cli\u003eDatta A, David R, Glennie S, Scott D, Cernuda-Morollon E, Lechler RI, Ridley AJ, Marelli-Berg FM (2006) Differential effects of immunosuppressive drugs on T-cell motility. American Journal of Transplantation 6:2871\u0026ndash;2883\u003c/li\u003e\n\u003cli\u003eDiehl R, Ferrara F, M\u0026uuml;ller C, Dreyer AY, McLeod DD, Fricke S, Boltze J (2016) Immunosuppression for in vivo research: state-of-the-art protocols and experimental approaches. Cellular \u0026amp; Molecular Immunology 2017 14:2 14:146\u0026ndash;179\u003c/li\u003e\n\u003cli\u003eHou FX, Yang HF, Yu T, Chen W (2007) The immunosuppressive effects of 10 mg/kg cyclophosphamide in Wistar rats. Environ Toxicol Pharmacol 24:30\u0026ndash;36\u003c/li\u003e\n\u003cli\u003eAn improved and versatile immunosuppression protocol for the development of tumor xenograft in mice - PubMed. https://pubmed.ncbi.nlm.nih.gov/25503146/. Accessed 3 May 2025\u003c/li\u003e\n\u003cli\u003eOkoli UA, Okafor michael T, Agu KA, et al (2020) Methodology for processing mastectomy and cryopreservation of breast cancer tissue in a resource- poor setting: A pilot study. Cryobiology 97:179\u0026ndash;184\u003c/li\u003e\n\u003cli\u003eLange T, Oh-Hohenhorst SJ, Joosse SA, et al (2018) Development and Characterization of a Spontaneously Metastatic Patient-Derived Xenograft Model of Human Prostate Cancer. Scientific Reports 2018 8:1 8:1\u0026ndash;11\u003c/li\u003e\n\u003cli\u003eMatossian MD, Burks HE, Elliott S, et al (2019) Drug resistance profiling of a new triple negative breast cancer patient-derived xenograft model. BMC Cancer 19:1\u0026ndash;17\u003c/li\u003e\n\u003cli\u003ePaez-Ribes M, Man S, Xu P, Kerbel RS (2016) Development of Patient Derived Xenograft Models of Overt Spontaneous Breast Cancer Metastasis: A Cautionary Note. PLoS One 11:e0158034\u003c/li\u003e\n\u003cli\u003eZhang X, Claerhout S, Prat A, et al (2013) A renewable tissue resource of phenotypically stable, biologically and ethnically diverse, patient-derived human breast cancer xenograft models. Cancer Res 73:4885\u0026ndash;4897\u003c/li\u003e\n\u003cli\u003eHidalgo M, Amant F, Biankin A V., et al (2014) Patient-derived Xenograft models: An emerging platform for translational cancer research. Cancer Discov 4:998\u0026ndash;1013\u003c/li\u003e\n\u003cli\u003eDerose YS, Wang G, Lin YC, et al (2011) Tumor grafts derived from women with breast cancer authentically reflect tumor pathology, growth, metastasis and disease outcomes. Nature Medicine 2011 17:11 17:1514\u0026ndash;1520\u003c/li\u003e\n\u003cli\u003eEirew P, Steif A, Khattra J, et al (2014) Dynamics of genomic clones in breast cancer patient xenografts at single-cell resolution. Nature 2014 518:7539 518:422\u0026ndash;426\u003c/li\u003e\n\u003cli\u003eChen B, Wei W, Huang X, Xie X, Kong Y, Dai D, Yang L, Wang J, Tang H, Xie X (2018) Circepsti1 as a prognostic marker and mediator of triple-negative breast cancer progression. Theranostics 8:4003\u0026ndash;4015\u003c/li\u003e\n\u003cli\u003eTang H, Huang X, Wang J, Yang L, Kong Y, Gao G, Zhang L, Chen ZS, Xie X (2019) CircKIF4A acts as a prognostic factor and mediator to regulate the progression of triple-negative breast cancer. Mol Cancer. https://doi.org/10.1186/s12943-019-0946-x\u003c/li\u003e\n\u003cli\u003eDe Neergaard M, Kim J, Villadsen R, Fridriksdottir AJ, Rank F, Timmermans-Wielenga V, Langer\u0026oslash;d A, B\u0026oslash;rresen-Dale AL, Petersen OW, R\u0026oslash;nnov-Jessen L (2010) Epithelial-stromal interaction 1 (EPSTI1) substitutes for peritumoral fibroblasts in the tumor microenvironment. American Journal of Pathology 176:1229\u0026ndash;1240\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003e\u003cstrong\u003eTable 2:\u003c/strong\u003e Clinical Signs for Toxicity Observation\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"700\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 48px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime Point\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTreatment Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003ePosture\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 87px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eLocomotion\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 76px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFur Condition\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eRespiration\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 84px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eEye/Nose Discharge\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 79px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eFeeding Behaviour\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 88px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eOverall Appearance\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003e30 min\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eCYA+Keto / CyX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003e4 hr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eCYA+Keto / CyX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003e24 hr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eCYA+Keto / CyX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003e48 hr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eCYA+Keto-treated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eMild change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003eMild change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eMild change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eMild change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eMild change\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003e48 hr\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eCyX-treated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eMild change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003eMild change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eMild change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eMild change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eMild change\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003eDay 7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eCYA+Keto-treated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eModerate change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003eModerate change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eModerate change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eModerate change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eModerate change\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003eDay 7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eCyX-treated\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eMild change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003eMild change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eMild change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eMild change\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eMild change\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"bottom\" style=\"width: 48px;\"\u003e\n \u003cp\u003eDay 14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eCYA+Keto / CyX\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 87px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 76px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 84px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 79px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 88px;\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eTable 2: Staged safe mouse pharmacological immunosuppression regimen for xenografts\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"652\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTime\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDose\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 274px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMode of Administration\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eDay 0\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003e17 mg/kg Ciprofloxacin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 274px;\"\u003e\n \u003cp\u003e\u0026nbsp;Gavage\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eDay1-5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003e35 mg/kg Cyclosporin A + 10 mg/kg Ketoconazole\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 274px;\"\u003e\n \u003cp\u003eCyclosporin A via intraperitoneal (i.p.) injection.\u003c/p\u003e\n \u003cp\u003eKetoconazole by oral gavage.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eDay 6 and Day 8\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 246px;\"\u003e\n \u003cp\u003e100 mg/kg cyclophosphamide\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 274px;\"\u003e\n \u003cp\u003eSubcutaneous injection\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eDay 9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 520px;\"\u003e\n \u003cp\u003eEstablished pharmacological immunosuppressed mice ready for xenograft\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6913154/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6913154/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eGenetically immunodeficient mouse models such as NOD/SCID and nude strains are widely used in cancer research for establishing patient-derived xenografts (PDXs). However, these models are expensive, require specialized facilities, and do not adequately reflect human immune-tumour interactions. This study aimed to develop a cost-effective and immunologically relevant pharmacological immunosuppression protocol in immunocompetent mice for the engraftment of triple-negative breast cancer (TNBC) xenografts in low-resource settings.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eFemale albino mice were treated with either cyclosporin A (35 mg/kg) plus ketoconazole (10 mg/kg) daily for five days, or cyclophosphamide (100 mg/kg) on alternate days (Days 0, 2, and 4). Haematological parameters, body weight, spleen weight, and feeding behaviour were monitored to assess immunosuppression and toxicity. A combined staged protocol was subsequently developed: cyclosporin A\u0026thinsp;+\u0026thinsp;ketoconazole for 5 days followed by cyclophosphamide (200 mg/kg on Day 6 and 100 mg/kg on Day 8), with orthotopic TNBC tissue implantation on Day 9.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCyclosporin A\u0026thinsp;+\u0026thinsp;ketoconazole significantly reduced total white blood cell, and lymphocyte counts by 65% and 75%, respectively (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and P\u0026thinsp;\u0026lt;\u0026thinsp;0.01). Cyclophosphamide alone induced a 50% lymphocyte reduction with minimal toxicity. The combined protocol enabled a 30% tumour take rate at first passage (P0) and 80% at second passage (P1). Recapitulates equal expression of Molecular analysis confirmed over 80% similarity between xenografts and the primary tumour.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis is the first article which demonstrates breast cancer patient derived xenograft in Nigeria and a pharmacologically induced immunosuppression regimen can support effective TNBC xenograft establishment in immunocompetent mice. The protocol is robust, cost-effective, and more accessible than genetically modified models, making it well-suited for use in resource-limited research settings.\u003c/p\u003e","manuscriptTitle":"Robust and Cost-Effective Pharmacological Immunosuppression Enables Establishment of Nigerian Patient-Derived Xenograft (PDX) Models of Triple-Negative Breast Cancer (TNBC) in Low-Resource Settings","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-06-24 12:39:45","doi":"10.21203/rs.3.rs-6913154/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3dd06a49-6543-4fc3-8f4f-b9e6f2fd722b","owner":[],"postedDate":"June 24th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":50351925,"name":"Biological sciences/Biochemistry"},{"id":50351926,"name":"Biological sciences/Biological techniques"},{"id":50351927,"name":"Biological sciences/Cancer"},{"id":50351928,"name":"Biological sciences/Molecular biology"},{"id":50351929,"name":"Health sciences/Diseases"},{"id":50351930,"name":"Health sciences/Medical research"},{"id":50351931,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2025-06-24T12:39:45+00:00","versionOfRecord":[],"versionCreatedAt":"2025-06-24 12:39:45","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6913154","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6913154","identity":"rs-6913154","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}