RETRACTED ARTICLE: Human induced pluripotent stem cell-derived platelets loaded with lapatinib effectively target HER2+ breast cancer metastasis to the brain

Prognosis of patients with HER2+ breast-to-brain-metastasis (BBM) is dismal even after current standard-of-care treatments, including surgical resection, whole-brain radiation, and systemic chemotherapy. Radiation and systemic chemotherapies can also induce cytotoxicity, leading to significant side effects. Studies indicate that donor-derived platelets can serve as immune-compatible drug carriers that interact with and deliver drugs to cancer cells with fewer side effects, making them a promising therapeutic option with enhanced antitumor activity. Moreover, human induced pluripotent stem cells (hiPSCs) provide a potentially renewable source of clinical-grade transfusable platelets that can be drug-loaded to complement the supply of donor-derived platelets. Here, we describe methods for ex vivo generation of megakaryocytes (MKs) and functional platelets from hiPSCs (hiPSC-platelets) in a scalable fashion. We then loaded hiPSC-platelets with lapatinib and infused them into BBM tumor-bearing NOD/SCID mouse models. Such treatment significantly increased intracellular lapatinib accumulation in BBMs in vivo, potentially via tumor cell-induced activation/aggregation. Lapatinib-loaded hiPSC-platelets exhibited normal morphology and function and released lapatinib pH-dependently. Importantly, lapatinib delivery to BBM cells via hiPSC-platelets inhibited tumor growth and prolonged survival of tumor-bearing mice. Overall, use of lapatinib-loaded hiPSC-platelets effectively reduced adverse effects of free lapatinib and enhanced its therapeutic efficacy, suggesting that they represent a novel means to deliver chemotherapeutic drugs as treatment for BBM.

Platelets are anucleate, cellular fragments derived from megakaryocyte membranes predominantly present in bone marrow [34][35][36][37] .Novel drug delivery systems have been designed to mimic various properties possessed by platelets, such as their ability to adhere to and deliver toxic drugs to tumor cells 38,39 .Most of these types of delivery systems utilize platelet membranes and require a complex production process.Platelet membrane-cloaked nanoparticles have also been considered but are still less biocompatible as they induce an immune response in vivo [40][41][42][43] .Unlike nanoparticle-based drug delivery systems, platelets are naturally cleared from the body by reticuloendothelial cells in liver and spleen [44][45][46][47][48][49] .Platelets harboring encapsulated agents have demonstrated systemic clearance similar to donor-derived platelets 28,39 .Previous studies report that platelets are activated by tumor cells and adhere to tumor cells, a phenomenon referred to as tumor cell-induced platelet aggregation (TCIPA), which can also increase metastasis 50 .Activated platelets release contents from their granules, and platelets loaded with drugs (such as doxorubicin 51 ) release those drugs in the proximity of tumor site 50 .However, cancer patients undergoing chemotherapy can be thrombocytopenic, and cancer patients who receive multiple allogenic drug-loaded platelet transfusions could develop refractoriness to platelets due to HLA alloreactivity and subsequently require transfusions with HLA-matched donor platelets loaded with drugs [52][53][54] .Finding alternative sources of non-immunogenic, high-quality autologous platelets could reduce these risks.
Since human induced pluripotent stem cells (hiPSCs) are a potentially replenishable source of transfusable drug-loaded HLA negative (Universal) or autologous or HLA matched platelets, our primary objectives were to develop a clinically adaptable protocol to generate hiPSC-derived megakaryocytes and platelets in vitro and then determine whether those hiPSC-platelets could serve as drug carriers to target HER2+ BBMs in vitro and in mouse models.In vivo, lapatinib-loaded hiPSC-platelets targeted BBMs and achieved a longer retention time than synthetic drug delivery systems or free drugs.Encapsulated lapatinib appeared to escape immunosurveillance and delivered drug in the vicinity of tumor cells via TCIPA, apparently circumventing the blood-brain barrier (BBB), which can interfere with treatment of brain metastases 55 .We conclude that drug-loaded hiPSC-platelets exhibit enhanced therapeutic efficacy at potentially reduced drug dosages, limiting damage to normal tissues, and could serve as an efficient drug carrier to treat HER2+ BBM tumors in brain.
Culturing of Day 0 immature megakaryocytes (Supplementary Fig. S2A (Gating Strategy)) in STEMspan Megakaryocyte Expansion Supplement (STEMCELL Technologies) and STEMspan ACF medium, along with addition of barasertib starting on Day 2, increased the ploidy of immature CD41a + CD42b + megakaryocytes (Supplementary Fig. S2A (Gating Strategy)) by Day 4 (Supplementary Fig. S1A (Scheme) and D), which was used as a surrogate marker for megakaryocyte maturation (" ≥ 8n ploidy" served as a marker of maturation).Addition of barasertib has been previously shown to promote generation of polyploid megakaryocytes (Supplementary ◂

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Fig. S1D and S2B) and increased terminal differentiation to matured megakaryocytes to platelets 64 .Similarly, megakaryocytes cultured in ultra-low adherent culture plates along with barasertib produced ~ 30 Calcein Blue AM + Annexin-V -CD41a + CD42b + platelets per megakaryocyte cell by day 7 (Fig. 1C,D) (Fig. 1C, Supplementary Fig. S2C), indicating that our protocol can successfully differentiate hiPSC cells through the stages of immature diploid megakaryocytes (Supplementary Fig. S2A), to maturation (Supplementary Fig. S2B) and to terminal differentiation of platelets (Supplementary Fig. S2C).
Large, highly polyploid (Supplementary Fig. S1D) megakaryocytes (Supplementary Fig. S1E) contained demarcation membrane systems and organelles, including mitochondria, granules, and multiple nuclei, and were capable of generating distinct pro-platelets (Supplementary Fig. S1G) by culture days 5-7 (Fig. 1D).Platelets can be damaged by extracellular metalloproteinases, which shorten their time in circulation and promote loss of surface CD42b expression relative to CD41 (αIIb) 65,66 .To determine if pro-platelets were differentiated and intact, we examined CD41a and CD42b expression in living (Calcein Blue AM + ) and non-apoptotic (Annexin V − ) hiPSC platelets purified by BSA gradient segregation (Fig. 1C,D and Supplementary Fig. S2C) and compared expression to infused donor platelets (Supplementary Fig. S2C-E).We observed no or minimal loss of surface CD42b on iPS-derived relative to donor platelets.Approximately 80% of CD41 + hiPSC-derived platelets were CD42b + .Another ~ 10% of CD41 + hiPSC-derived platelets were smaller in size and granularity relative to the larger proportion of CD41 + CD42b + hiPSC-derived platelets.We conclude that this 10% was non-functional and did not count them as platelets (Supplementary Fig. S2E).Using platelets from human blood donors to establish size gating (Fig. 1C and Supplementary Fig. S2C), we observed that the ~ 80% of hiPSC-platelets generated above expressed CD41a and CD42b comparably to donor platelets.We then examined the kinetics of platelet production from maturing megakaryocytes by flow cytometry-based counting of CD41 + CD42b + platelet-sized particles and found that platelet levels began to increase on day 5 and peaked at day 7 (Fig. 1D).
Transmission electron micrography (TEM) analysis demonstrated that hiPSC-platelets were ultrastructurally identical to human donor platelets (Fig. 1E): hiPSC-platelets were anucleate and possessed organelles seen in adult human blood donor platelets, including mitochondria, OCSs (open canalicular systems), and granules.We then performed light transmission aggregometry (LTA), a method used clinically to evaluate platelet function in vitro [67][68][69] , on hiPSC-platelets generated in vitro from hiPSCs and showed that 3 × 10 7 resting hiPSC-platelets resuspended in human plasma reached 50-60% aggregation at all time points analyzed after exposure to 20 µM ADP and TRAP (Fig. 1F).Donor platelets exhibited stronger aggregation and reached 85-80% aggregation under the same conditions.To assess platelet activation, we performed activation assays using a FITC-conjugated PAC1 antibody, which binds to the activated conformation of αIIbβ3 integrin, and a PE-conjugated P-Selectin monoclonal antibody, which binds to P-selectin (P-Sel) on the surface of granules in activated platelets.In response to ADP and TRAP treatment, hiPSC-platelets showed stronger PAC1 binding than unstimulated controls (Fig. 1G,H and Supplementary Fig. S3A).PAC1 and P-Sel activation profiles of hiPSC-platelets were similar to those of donor platelets, although some hiPSC-platelets showed pre-activation (Fig. 1G,H), as has been reported by others 66 .

Functional comparison of lapatinib-loaded and non-loaded hiPSC-derived platelets.
We next confirmed that our hiPSC-platelets could encapsulate drugs by assessing uptake of the fluorescent probe coumarin-6 (C6, 0.1%) by fluorescence imaging (Fig. 2A).We then demonstrated that doxorubicin could be encapsulated into hiPSC-derived platelets using the same protocol, suggesting that lapatinib could be similarly encapsulated (Supplementary Fig. S4A).We and others 51,[70][71][72] previously demonstrated that donor platelets can take up drug via passive transfer (Supplementary Fig. S4A).We calculated the encapsulation efficiency (a number that describes the extent of drug encapsulation) to be 93% in hiPSC-platelets, while drug loading capability (which indicates the amount of drug loaded per unit weight of hiPSC-platelets) to be 49% (Fig. 2B), the latter suggesting that the volume ratio of hiPSC-platelets to lapatinib was ~ 1:2.Interestingly, pH modulated lapatinib release in vitro: release was slower at pH 7.4 but more rapid at pH 6.5.Accordingly, at pH 6.5, ~ 80% of loaded lapatinib was released into the buffer within 40 h, as compared to ~ 40% release at pH 7.4 within 40 h (Fig. 2C).
Additional TEM analysis indicated that both lapatinib-loaded and non-loaded platelets showed organelles such as mitochondria, granules and glycogen granules, and that lapatinib-loaded platelets exhibited normal morphology (Fig. 2D).Lapatinib-loaded hiPSC-platelets and donor platelets showed comparable activation capacity in vitro based on surface expression of PAC1 and P-Sel (Fig. 2F and Supplementary Fig. S3A), as well as a similar ability to aggregate in vitro after ADP and TRAP exposure (Fig. 2E).These analyses confirm that hiPSC-platelets maintain integrity and biological function after lapatinib loading and retain properties of bona fide human platelets.

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V levels, while exposure to non-loaded controls had little or no effect Annexin-V positivity (Fig. 3C,D).RT-qPCR analysis of FACS-sorted CD326 + BBM cells revealed significant upregulation of the pro-apoptotic genes BAD, BAK, BAX, and p53 in BBM cells exposed to lapatinib-loaded hiPSC-platelets relative to those exposed to non-loaded control hiPSC-platelets (Fig. 3E), indicative of apoptosis.To determine whether BBM cells contained lapatinib delivered by hiPSC-platelets, we first loaded hiPSC-platelets with saline, the fluorescent probe C6, or a 1:1 mixture of lapatinib and C6 and analyzed C6 accumulation in BBM cells by flow cytometry (Fig. 3F and Supplementary Fig. S5A).We then isolated C6-positive BBM cells by FACS and analyzed lapatinib concentrations in these cells via LC-MS/MS (Supplementary Fig. S5B) as well as viability using CCK8 assays (Fig. 3G).Lapatinib levels were significantly elevated in BBM cells exposed to lapatinib-loaded hiPSC-or donor-platelets compared to BBM cells exposed to C6 only-loaded hiPSC-or donor-platelets or with non-loaded platelets (Controls) (Fig. 3F,G and Supplementary Fig. S5A and S5B).We also observed significantly reduced viability of BBM cells exposed to a 1:1 mixture of lapatinib and C6, as compared to non-loaded or C6-loaded platelets (Fig. 3G).
Analysis of therapeutic efficacy of lapatinib-loaded hiPSC-platelets in vivo.We next assessed potential anti-tumor effects of lapatinib-loaded hiPSC-platelets in vivo using female NOD/SCID mice bearing xenograft BBM tumors, which we developed 41,74 (Fig. 4A).To do so, we injected BBM cells (100 K) expressing ZsGreen1 Renilla luciferase intracranially in the subcortical region of mice via the cisterna magna on day 0 and allowed tumor development to occur over 7 days.Once tumors were detectable by bioluminescence imaging (BLI) lapatinib-loaded hiPSC or donor platelets, free lapatinib, or non-loaded control hiPSC and donor platelets were intravenously administered via the tail vein every 3 days.Tumor progression was then monitored by BLI every 2 days up to day 30 (Fig. 4A,B,E).That analysis revealed significantly reduced BLI counts in mice infused with lapatinib-loaded hiPSC-or donor-platelets relative to cohorts that received non-loaded hiPSC-or donor-platelets or free lapatinib (Fig. 4A,B).We monitored mice for up to 60 days and observed that BLI intensity decreased to undetectable levels in mice treated with lapatinib-loaded hiPSC-platelets (Fig. 4A) relative to vehicle-or non-loaded platelet-treated controls.Moreover, overall survival of mice bearing BBM tumors was significantly higher in animals treated with lapatinib-loaded hiPSC-or donor-platelets relative to vehicle-or non-loaded platelet-treated controls (Fig. 4C,E and Supplementary Fig. S6B and C).Pre-treatment of BBM1 cells with lapatinib-loaded hiPSC-platelets also reduced tumor seeding capacity of BBM1 cells by 1,000-fold relative pre-treatment with non-loaded hiPSC-platelets (Fig. 4D).As expected, tumor size (as determined using HEstained horizontal brain sections and AMIRA-based area quantification) also decreased significantly in tumorbearing mice infused with lapatinib-loaded hiPSC-or donor-platelets relative to free lapatinib or untreated controls (Fig. 4E and Supplementary Fig. S7C).Also, after 24 days of treatment with free lapatinib, mice had a final average body weight of ~ 18.9 g, indicative of significant weight loss, compared to animals treated with non-loaded or lapatinib-loaded hiPSC-platelets (Fig. 5A), suggesting that lapatinib toxicity to normal tissue is reduced by encapsulation in hiPSC-or donor-platelets.Interestingly, histopathological analysis using hematoxylin and eosin (H&E) staining of heart, liver, spleen, lung, and kidney did not reveal significant abnormalities in any of the treatment groups (Supplementary Fig. S6A).
We also measured plasma lapatinib concentrations in mice bearing BBM derived tumors at various timepoints after treatment (Fig. 5B).Whereas free lapatinib was rapidly cleared with a short half-life (t 1/2 = 2.1 ± 0.6 h), lapatinib released from lapatinib-loaded hiPSC-or donor platelets remained at a higher level with a longer half-life (t 1/2 = 31.3± 0.9 h; Fig. 5B).Analysis of tissue distribution showed that, compared to levels of lapatinib released from lapatinib-loaded hiPSC-or donor-platelets, concentrations of free lapatinib were significantly higher in heart, kidney and liver tissues but relatively lower in tumor tissue (Fig. 5C).However, we observed an increase in lapatinib concentration in lungs of tumor-bearing mice treated with lapatinib-loaded hiPSC-and donor-platelets relative to free lapatinib.Furthermore, lapatinib levels were higher in kidney and heart in free lapatinib-administered animals relative to animals treated with lapatinib-loaded hiPSC-and donor-derived platelets, suggesting decreased toxicity (Fig. 5C).To confirm minimal adverse effects in animals treated with lapatinib-loaded donor-or hiPSC-platelets, we performed histopathological analyses of all major organs including lung, kidney, and heart from BBM tumor-bearing mice treated with both.Relative to non-loaded platelet controls, we observed no significant changes in morphology in any group analyzed (Supplementary Fig. S6A).To confirm that elevated lapatinib levels did not alter heart function we also quantified levels of cardiac troponin I (cTnI) in sera of variously treated tumor-bearing mice and found that levels were lower in all test groups than those seen in positive control mice exposed to isoproterenol, which induces higher troponin I levels, strongly suggesting that heart function is normal in mice treated with lapatinib-loaded or non-loaded donor or hiPSCplatelets (Supplementary Fig. S7A).Circulation or clearance kinetics of 5 × 10 8 intravenously infused non-loaded or lapatinib-loaded human iPSC-and donor-platelets was comparable in macrophage-depleted NOD/SCID mice, with a time to reach maximal accumulation (Tmax) of 1 h (Supplementary Fig. S7B and S8A-B).HiPSC-platelets, like human blood platelets, circulated for at least 24-48 h, indicating that lapatinib-loaded hiPSC-platelets possess a lower rate of clearance from blood circulation, which may allow them to accumulate and release lapatinib in the vicinity of the tumor tissues, potentially sparing normal tissues.

Discussion
Breast cancer patients can develop BBMs years or even decades after their initial diagnosis, indicative of a long latency period, despite the presence of circulating tumor cells 12,75,76 .Metastases are responsible for 90% of all cancer deaths 3,4,7,8 .Patients diagnosed with brain metastases have a dismal probability of 1-year survival 1 .Furthermore, the advancement and improved efficacy of treatments for primary breast cancer have led to an increased propensity to develop metastatic breast tumors 6,10 .Although all breast cancer subtypes can metastasize to the brain, patients with HER2+ primary breast tumors have a higher risk of developing brain metastasis 9 .~ 40% www.nature.com/scientificreports/ of patients with HER2+ primary breast cancer develops brain metastasis 1,6 .Major treatment modalities for HER2+ BBMs have been radiation therapy, surgical resection of BBM tumors, and systemic chemotherapy treatment with various drugs [77][78][79][80][81][82] .However, the benefits of radiation therapy are limited to short-term palliation of distressing symptoms and this modality cause neurocognitive dysfunction 83 .Additionally, a major limitation in developing effective chemotherapeutic treatments for HER2+ BBMs is the poor activity of anti-neoplastic agents against HER2+ BBMs and poor penetration of these agents into brain tissue 3,7,8 .Previous studies reported modest activity of lapatinib against BBM tumors in the brain 22,[84][85][86] .Therefore, development of drug delivery systems that can augment BBB clearance to reach the tumors residing in the brain, reduce side effects and simultaneously improve the efficacy, therapeutic index, and biocompatibility of promising drugs treatments is needed [27][28][29][30]33,42,87 . Althoughvarious drug delivery systems have been developed, poor biocompatibility has limited their application in clinical settings.Some studies have demonstrated the utility of natural donor-derived blood platelets as drug carriers 51 .For example, a recent study demonstrated the use of doxorubicin-loaded blood-platelets as a delivery system to treat lymphoma and achieved a longer retention time, relative to free drug retention times.However, allogenic drug-loaded platelet transfusions run the risk of alloimmunization to HLA 52,53,61 .Patients who receive multiple platelet transfusions, such as those with various types of cancer, often develop platelet refractoriness due to HLA alloreactivity and subsequently require additional transfusions with HLA-matched donor platelets [88][89][90][91] .Therefore, finding alternative sources of non-immunogenic, high-quality platelets might decrease risks associated with allogeneic drug-loaded platelet transfusions.

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Therefore, our main objectives were to develop a clinically adaptable protocol to generate hiPSC-derived megakaryocytes that can efficiently mature and terminally differentiate into highly functional platelets and to determine whether hiPSC-platelets possess properties of natural platelets and could serve as an alternative to allogeneic donor-derived platelets as drug carriers for targeting and treating BBMs.Here, we report a serum and feeder-free protocol to differentiate human hiPSCs into megakaryocytes, which in turn mature and terminally differentiate to generate functional platelets.Using this method, we generated hiPSC-platelets on a large scale, allowing us to perform flow cytometry-based activation assays and LTA-based aggregation assays, in vitro.The latter demonstrated that donor-derived platelets do aggregate more readily than hiPSC-derived platelets, potentially due to the method used to enrich them.Specifically, we performed differential centrifugation to remove the naïve megakaryocytes from platelet medium followed by BSA density gradient-based centrifugation to generate purer platelet populations.These methods efficiently remove large megakaryocytes but cannot remove small-sized debris materials (e.g., CD42b -or CD41a -or Annexin-V + particles) that does not aggregate and can lead to higher background.This purification step does not occur in the preparation of fresh donor platelets.Furthermore, hiPSC derived platelets are fetal in nature showed better aggregation profiles compared to platelets derived from CD34+ umbilical cord blood cells, which show no or minimal aggregation based on LTA representing significant improvement 61 .Subsequently, we used these hiPSC-platelets to encapsulate lapatinib and found that they can be loaded with an encapsulation efficiency of ~ 90% without any alteration in platelet morphology and functionality (Fig. 2).Using Boyden chambers, we observed that lapatinib-loaded hiPSC-and donor-derived platelets significantly reduced the viability of BBM1 and BBM2 cells compared to free lapatinib (Fig. 3B).We attribute this increased cytotoxicity to the rapid migration of the lapatinib-loaded platelets from the top chamber to the bottom chamber, where the BBM1 and BBM2 tumor cells were cultured in a monolayer; however, this remains to be validated through time-lapse imaging based analyses of binding kinetics of variously stained platelets binding to the tumor cells.
We also showed that the in vitro release of lapatinib from lapatinib-loaded hiPSC-and donor-derived platelets was pH-sensitive.This is critical, as the tumor microenvironment is acidic compared to normal tissues, reflecting the hypoxic conditions induced by rapid tumor cell proliferation 92,93 .However, it remains unclear how lapatinib release from hiPSC-and donor-derived platelets is accelerated under acidic conditions.The lower pH could induce the generation of platelet-derived extracellular vesicles containing lapatinib, as has been reported for cancer cell-derived vesicles 94 .Loaded platelets can also respond to thrombin-mediated activation and generate extracellular vesicles, which could augment lapatinib release.Such vesicles, which are ~ 200 nm in diameter, could be important secondary drug delivery systems for lapatinib with greater potential to infiltrate BBM tumors and fuse with cancer cells.In addition, an acidic environment can promote the generation of mixed platelet-leukocyte aggregates and increased chemotaxis of neutrophils mediated by platelets in a P-Sel-dependent manner 95 ; and P-Sel, in turn, is exposed on the platelet surface under acidic conditions.Finally, alterations in platelet structure have been reported under acidic conditions 96 , which may increase OCS "permeability" 97 .Overall, these findings suggest that drug-loaded hiPSC-and donor-derived platelets are more likely to release their contents in or around the acidic environment established by BBM tumor cells rather than the normal physiological environment, reducing potential toxicity to surrounding tissues.
After treatment with free lapatinib and lapatinib-loaded hiPSC-platelets, the growth inhibition and apoptosis of BBM cells were comparable in vitro.However, the effective treatment of brain metastases is generally hindered by the BBB 55 .Thus, our more clinically significant finding was that hiPSC-platelets can act as an efficient drug carrier for the treatment of HER2+ BBMs tumors residing in the brain.We found that, compared to the infusion of free lapatinib, the infusion of lapatinib-loaded hiPSC-platelets significantly decreased tumor progression and tumor size and more potently reduced the tumor-seeding capability of BBM cells.Plasma analyses also indicated that lapatinib concentrations remained higher in mice treated with lapatinib-loaded hiPSC-platelets, with a much longer half-life than the free drug.These results suggest that lapatinib-loaded hiPSC-platelets could be more effective against BBM than free lapatinib.Furthermore, following infusion of lapatinib-loaded hiPSC-platelets, lapatinib levels were significantly higher in tumors relative to the major organs evaluated, which was not the case in mice infused with free lapatinib.Consistent with these findings, our histological assessment of various tissues in treated mice revealed no overt signs of injury; however, we did observe body weight loss in tumor-bearing mice treated with free lapatinib, which is an early indicator of toxicity.Finally, cTnI levels were low and comparable

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Breast-to-brain metastasis (BBM) cell cultures.BBM tissue specimens (HER2+) were collected and propagated as previously described 73 .Briefly, the cell lines COH-BBM1 (BBM1) and COH-BBM2 (BBM2) were established by validation of phenotypic markers (HER2, pan-cytokeratin) and exclusion of brain cells (astrocytes, microglia, and endothelial cells), and cultured in DMEM/F12 media (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Sigma Aldrich), 1% Glutamax (Life Technologies), and 1% antibiotic-antimycotic (Life Technologies), at 37 °C and 5% CO 2 .Cell line authentication was performed by short tandem repeat profiling by IDEXX BioAnalytics, and the cell lines were determined to be Mycoplasma-negative by PCR (Agilent Mycosenser Mycoplasma Assay Kit) as recently as 1 month before the final experiments.
In vivo treatment of BBM xenograft tumors with free lapatinib and lapatinib-loaded hiPSC-platelets.All NOD/SCID mice were maintained under veterinary supervision and housed under standard living conditions, with a 12-h light/dark cycle and access to food and water ad libitium.Investigators were blinded to treatment group for the analysis of mice.The experiments were not randomized.No statistical methods were used to predetermine sample size.No strategy was used to eliminate or identify confounders.Female NOD/SCID mice were randomized to treatment and control groups of n = 7, giving 80% power to detect a treatment effect size of 65% compared to a baseline response of 5% at a significance level of 0.05.Throughout the course of our experiments, no animals were excluded from the study.Macrophages were depleted in all NOD/SCID mice by intravenous injection of liposome-encapsulated clodronate, as shown previously 61,99 .
To evaluate tumor growth and survival in vivo, BBM1 cells (100 K in 20 μL PBS) were transduced with mCherry and firefly luciferase (mCherry:LUC, Addgene) [100][101][102] and injected into the brains of 6-week-old NOD/ SCID female mice (7 mice/group, 24 total; 2 mm right and 1 mm anterior to the bregma suture).Only NOD/ SCID female mice were used for these studies because BBM tumors occur predominantly in females.Tumor progression was monitored every 48 h for 40 days using BLI on a Xenogen Imaging System (Xenogen Corp).At the conclusion of the experiments, mice were euthanized, and their brains and other tissues collected and fixed in formalin (Thermo Fisher Scientific) for downstream lapatinib concentration analyses.
The tumor-bearing female NOD/SCID mice were randomly divided into six groups to receive treatment with: (1) normal saline; (2) non-loaded hiPSC-platelets; (3) free lapatinib; (4) lapatinib-loaded hiPSC-platelets; (5) non-loaded donor-derived platelets; (6) lapatinib-loaded donor-derived platelets.Each group had n = 7 female NOD/SCID mice, for a total of 42 animals.The dose of lapatinib for each injection was 6 mg/kg and based on the effective drug loading rate of 48.5%, the dose of lapatinib-loaded hiPSC-platelets was 12 mg/kg.Treatments were intravenously administered via the tail vein, and the volumes of tumors were measured every 3 days.

Statistical analyses.
Data are presented as mean ± standard deviation (SD), using data generated from n = 3 biological replicates with n = 2 technical replicates present for each biological replicate.The statistical significance of differences between groups was determined (unless otherwise noted) using one-or two-way analysis of variance (ANOVA) with Bonferroni correction for multiple comparisons (GraphPad Prism 8.