---
title: "Human platelet lysate supports expansion of tumor-infiltrating lymphocytes in endometrial cancer"
id: "frontiers-in-immunology-19-human-platelet-lysate-enables-robust-expansion-of-tumor-infiltrating"
canonical_url: "https://medichelpline.com/clinical-feed/frontiers-in-immunology-19-human-platelet-lysate-enables-robust-expansion-of-tumor-infiltrating"
content_type: "clinical_feed_article"
specialty: "Oncology"
source_name: "Frontiers in Immunology"
source_url: "https://www.frontiersin.org/articles/10.3389/fimmu.2026.1873651"
published_at: "2026-10-14T00:00:00.000Z"
evidence_level: "Journal Feed"
license: "CC-BY-NC-4.0 / Informational Use"
---
# Human platelet lysate supports expansion of tumor-infiltrating lymphocytes in endometrial cancer
## Provenance & Clinical Metadata
- **Canonical URL:** https://medichelpline.com/clinical-feed/frontiers-in-immunology-19-human-platelet-lysate-enables-robust-expansion-of-tumor-infiltrating
- **Specialty:** [Oncology](https://medichelpline.com/clinical-feed/oncology.md)
- **Primary Source:** Frontiers in Immunology
- **Source URL:** [Original Journal Publication](https://www.frontiersin.org/articles/10.3389/fimmu.2026.1873651)
- **Published At:** 2026-10-14T00:00:00.000Z
- **Evidence Rating:** Journal Feed
## Executive GIST (TL;DR)
- The article title indicates **Human platelet lysate** (HPL) was used to expand **tumor infiltrating lymphocytes** (TILs) from **endometrial cancer** tissue. The source content provided here did not include experimental metrics or numerical outcomes. - No methods were available in the provided source: the report did not document culture conditions, HPL preparation, cell counts, or expansion timelines. - The source did not present immunophenotyping results, such as CD4/CD8 ratios, activation markers, or exhaustion markers for the expanded TILs. - Functional assays (cytotoxicity, cytokine release, antigen specificity) were not included in the captured content. - Safety, sterility, and reproducibility data — including lot-to-lot variability of HPL — were not reported in the excerpt available. - Clinical translation details (patient selection, tissue procurement, regulatory approvals, or planned clinical testing) were absent from the supplied source material. - Authors, institutional affiliations, and specific journal metadata beyond the article title and outlet were not supplied in the captured text. - Key unanswered items remain: exact experimental endpoints, quantitative expansion rates, viability and phenotype of TILs, and any comparative controls used; the source did not report these elements.
## Clinical Analysis & Structured Key Points
Frontiers | Human platelet lysate enables robust expansion of tumor infiltrating lymphocytes from endometrial cancer ORIGINAL RESEARCH article Front. Immunol. , 14 October 2026 Sec. Cancer Immunity and Immunotherapy Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1873651 Published in Frontiers in Immunology Cancer Immunity and Immunotherapy 7 impact factor 11.3 citescore Editor & Reviewers Edited by A B Amorette Barber Reviewed by S P Soumya Panigrahi E L Elisabetta Liverani Outline Figures and Tables Figure 1 View in article Figure 2 View in article Figure 3 View in article Figure 4 View in article Table 1 Characteristics of the patients who underwent hysterectomy and from whom the EC samples were obtained. View in article Table 2 Exhaustion scores post-REP TILs compared to CAR-T cells. View in article ORIGINAL RESEARCH article Front. Immunol. , 14 October 2026 Sec. Cancer Immunity and Immunotherapy Volume 17 - 2026 | https://doi.org/10.3389/fimmu.2026.1873651 Human platelet lysate enables robust expansion of tumor infiltrating lymphocytes from endometrial cancer W I Willemiek Iwema 1 † N V Nienke van Rooij 1 † L R Lena Rockstein 2 A P Annechien Plat 1 R Y Refika Yigit 1 J B Joost Bart 3,4 B G Bahez Gareb 5 H W Hans W. Nijman 1 ‡ E B Edwin Bremer 2 ‡ M D Marco de Bruyn 1 ‡ * 1. Department of Obstetrics and Gynaecology, University Medical Center Groningen, University of Groningen, Groningen, Netherlands 2. Department of Hematology, University Medical Center Groningen, University of Groningen, Groningen, Netherlands 3. Department of Pathology, University Medical Center Groningen, University of Groningen, Groningen, Netherlands 4. Department of Pathology, Leiden University Medical Center, Leiden, Netherlands 5. Department of Clinical Pharmacy and Pharmacology, University Medical Center Groningen, University of Groningen, Groningen, Netherlands See more Article metrics View details Abstract Background/objectives: Tumor infiltrating lymphocytes (TIL) therapy has yielded promising results in melanoma patients, with an approved product available. TIL therapy is of similar interest to a number of additional malignancies, including endometrial cancer (EC). For EC specifically, the tumor is well infiltrated with TILs and has accessible tumors as starting material for TIL isolation/expansion. However, the TIL manufacturing process currently relies on human serum (HS), which is difficult to source and has considerable batch-to-batch variation. Here, we explored the feasibility of using human platelet lysate (HPL) as a good manufacturing practice (GMP)-compatible alternative. Methods: EC material was obtained following surgery and used to expand TILs in pre–rapid expansion protocols (pre-REP) and REP phases, comparing HPL- and HS-containing media. Tumor tissue from 10 EC patients, representing both high and low tumor mutational burden histotypes was used. Spatial TIL infiltration was assessed using immunohistochemistry. After expansion, TIL phenotype was determined using flow cytometry for CD3, CD56, CD8, CD39, and CD103. TILs from the REP were further characterized with spectral flow cytometry using markers for differentiation, activation, co-stimulatory receptors, and immune checkpoints. Results: TILs expanded comparably using both HPL- and HS-media during pre-REP and REP in terms of cell yields. All tumors showed an excluded spatial immune phenotype. Immunophenotypes were consistent across media conditions. Spectral cytometry revealed patient-specific clustering unaffected by supplement type. Conclusions: TILs can be successfully expanded from EC using a fully GMP-compatible HPL protocol. Our findings may serve as a foundation for further improvements of existing GMP-compliant TIL manufacturing protocols. 1 Introduction Tumor infiltrating lymphocytes (TIL) therapy has emerged as a highly promising treatment in oncology, demonstrating encouraging results in solid tumors, such as melanoma, cervical cancer, renal cell carcinoma, and non-small cell lung cancer ( 1 – 5 ). In melanoma, TIL therapy was effective in patients who had progressed after treatment with immune checkpoint inhibitors (ICI) ( 4 ). Due to its clinical efficacy in melanoma, the first TIL therapy was approved by the FDA in 2024 for the treatment of unresectable or metastatic melanoma ( 4 ). Endometrial cancer (EC) is the most common gynecological malignancy in high-income countries and is characterized by a rising global incidence and mortality ( 6 ). For patients with advanced or recurrent microsatellite instability-high or mismatch repair-deficient EC, current standard treatment involves ICIs. However, as in melanoma, not all patients respond to ICI. Furthermore, in EC the tumor is well infiltrated with TILs and has accessible tumors to source starting material for TIL isolation, and consequently, EC represents a logical indication for TIL therapy ( 7 – 10 ). A TIL product is classified as an advanced therapy medicinal product (ATMP) and should therefore be manufactured under good manufacturing practice conditions (GMP). The manufacturing of TILs starts with a solid piece of tumor tissue obtained during surgery, and consists of two phases: pre-rapid expansion protocol (pre-REP), and the rapid expansion protocol (REP). The pre-REP phase involves ex vivo expansion of T cells through digestion of the tumor tissue and incubation with interleukin-2 (IL-2), with the aim of expanding tumor-reactive T cells. TILs are subsequently expanded to therapeutic cell numbers during the REP phase, in which pooled pre-REP TILs are cultured with anti-CD3 and IL-2, and activated using irradiated peripheral blood mononuclear cells (PBMC) feeder cells to drive expansion. During the manufacturing process, in-process control (IPC) and quality control tests are performed to warrant product quality and safety. Following a lymphodepleting chemotherapy regimen, the expanded TILs are administered intravenously to the patient, with concurrent IL-2 administrations to promote in vivo TIL expansion and enhance the antitumor activity. Current TIL manufacturing processes utilizes human serum (HS), which presents challenges related to limited availability and, more importantly, substantial batch-to-batch variation. For GMP compliance, raw materials with limited batch-to-batch variability are desired, ensuring a standardized quality and manufacturing process. To address these limitations, we investigated whether human platelet lysate (HPL) could effectively replace HS in TIL expansion protocols. HPL is more readily available, has reduced batch-to-batch variation when using large-scale standardized methods, and has shown comparable activity to HS in e.g. stem cell culture ( 11 – 13 ). Currently, HPL is available in clinical-grade and already used in clinical trials for CAR-T cell manufacturing under GMP conditions ( 14 – 16 ). We evaluated the feasibility of isolating and expanding TILs from EC tumor samples using HPL as a substitute for HS. The critical quality attributes as well as phenotypic properties were extensively characterized to assess the impact of HPL supplementation during the manufacturing process. 2 Materials and methods 2.1 Human material Patients were enrolled in a clinical protocol approved by the Institutional Medical Ethical Board. Informed consent was obtained from all patients before tissue collection. Tumor samples were acquired during a standard-of-care surgical procedure, and routine pathologic assessment confirmed all included tissue as endometrial cancer. Tissue collection occurred between June and September 2023 for TIL cultures. Allogeneic peripheral blood mononuclear cells, called feeder cells, were derived from apheresis of healthy donors at the Sanquin blood bank in the Netherlands. All donors underwent screening for infection markers, including hepatitis B, hepatitis C, hepatitis E, HIV, and syphilis (RPR). Apheresis samples were purified using Ficoll-Paque (Cytiva) density gradients and subsequently cryopreserved. 2.2 Pre-rapid expansion protocol TIL cultures were established according to previously described protocols ( 2 , 3 , 17 ) with modifications. Tumor tissue was aseptically excised during surgery. A portion of the tissue was transferred to a tube containing RPMI medium (Life Technologies) with 10% fetal bovine serum (FBS) (Capricorn Scientific). The tissue was dissected into small fragments in a laminar flow cabinet and transferred into a GentleMACS C-tube (Miltenyi) containing 5–10 ml digestion medium. Digestion medium consisted of TexMACS medium (Miltenyi) supplemented with 12,6 E/ml pulmozyme (Roche). The tissue suspension was dissociated using a GentleMACS Dissociator (Miltenyi). Program h_tumor_01 was run 3–4 times, and the tissue suspension was subsequently filtered through 70 µm cell strainers (Falcon) to remove undigested debris. A Ficoll density gradient was performed to enrich for lymphocytes. After washing with PBS (Apotheek A15), the cell digest was counted, and viability was assessed using a disposable C-Chip Neubauer improved counting chamber (NanoEnTek) with trypan blue (Sigma Aldrich) staining. The cells were plated in a 24-well tissue culture plate at a density of 1x10 6 total cells per well in 1 mL medium. The culture medium consisted of 90% TexMACS medium, 50 µg/ml gentamicin sulfate (Centrafarm), 6000 U/mL interleukin-2 (IL2) (Novartis), 10% human AB serum (HS; MP Biomedicals) or 10% human platelet lysate (HPL; ELAREM Perform 1 research grade; PL BioScience), and 0.6 U/ml heparin sulfate (Apotheek Albert Schweitzer ziekenhuis, Dordrecht) when using HPL. The plates were incubated in a humidified incubator at 37 °C with 5% CO 2 . Cultures were maintained for 2–4 weeks, with biweekly inspection under a microscope. Medium was replaced with fresh medium (50% change) once a week. When the well became too full, the culture was resuspended and split at a ratio of 1:1 to 1:2. When sufficient TIL growth was achieved, defined as multiple wells showing high cell density, indicated by the formation of lymphocyte clumps, the cultures were cryopreserved using 90% FBS and 10% DMSO and stored in liquid nitrogen till further use. TIL cultures were excluded for further analysis if there was less than 2 times fold expansion after 4 weeks of culturing. 2.3 Rapid expansion protocol The rapid expansion of pre-REP TIL cultures was performed using a previously described protocol ( 3 ). TILs were thawed one day before REP initiation with FBS and cultured in a 24-wells plate, using 10% HS or HPL medium supplemented with 6000 IU/ml IL2. On the day of REP initiation, feeder cells were thawed, resuspended in TexMACS medium supplemented with 50 µg/ml gentamicin sulfate, 6000 U/mL interleukin-2, 10% HS or HPL, and 0.6 U/ml heparin sulfate when using HPL. After irradiation at 40 Gy (IBL 637 Cesium-137y-ray machine), the feeder cells were mixed with TILs at a 1:200 TIL-to-feeder cell ratio, using 0.1x10 6 TILs. The TIL-feeder mixture was first cultured in T25 flasks, in an upright position for the first 7 days, with 20 ml medium containing 95% TexMACS medium with 5% HS or HPL, 50 µg/ml gentamicin, 3000 IU/ml IL-2 and 30 ng/ml anti-CD3 (OKT3; BD Biosciences). In the case of HPL medium, also 0.6 U/ml heparin sulfate was added. The cultures were incubated at 37 °C in 5% CO 2 for 14 days. Medium was changed on day 5 or 6, replacing 14 ml with fresh medium without OKT3. From day 5 or 6 onward, cells were checked every 2–3 days and split if necessary. After expansion, all samples were analyzed by flow cytometry and cryopreserved using FBS with 10% DMSO for future use. 2.4 Conventional flow cytometry After cryopreservation, TILs were thawed in FBS, washed with PBS 2% FBS, and incubated for 30 min on ice in the dark with the following antibodies: CD56 PE (eBioscience), CD8 APCeFluor780 (eBioscience), CD3 BV421 (BD Biosciences), CD4 PerCp Cy5.5 (eBioscience), CD39 APC (Thermo Fisher) and CD103 FITC (BD Biosciences). Samples were washed twice with PBS 2% FBS and kept on ice till analysis. Before analysis on the Agilent NovoCyte Quanteon flow cytometer, Propidium Iodide (PI, Sigma) staining was used to discriminate live/dead cells. UltraComp ebeads (Thermo Fisher) were used for compensation controls. The data was analyzed using Cytobank. 2.5 Spectral flow cytometry From TIL samples of different manufacturing conditions, 2x10 6 cells were collected, washed with and resuspended in 100 µL PBS. The viability marker LIVE/DEAD™ Fixable Blue dye (Thermo Fisher) was added (1 µL) and incubated for 20 min at room temperature (RT) in the dark. Samples were washed once with PBS + 5% FBS and blocked for 10 min at RT with 10 µL TruStain FcX, followed by an extra incubation of 10 min with 5 µL of CellBlox blocking buffer (Thermo Fisher) and TCRγδ antibody. After washing with PBS + 2% FBS, samples were stained for 10 min at 4 °C with 118.5 µl antibody mix ( Supplementary Table 1 ) containing 10 µL Brilliant Stain Buffer Plus (USUS Biosciences, San Jose, CA, USA). Following staining with the antibody mix, samples were washed three times with and resuspended in 200 µl PBS + 2% FBS. All washing steps were performed at 300×g for 5 min. An overview of all antibody clones, suppliers, and titrated volumes are indicated in Supplementary Table 1 . Samples were measured on a 5-laser Aurora spectral flow cytometer (Cytek Biosciences, Fremont, CA, USA) and data were analyzed using OMIQ flow cytometry software (Dotmatics, Boston, MA, USA). 2.6 Analysis spectral flow data Samples were imported into OMIQ and subjected to data cleanup using the FlowAI function with the following settings: Anomaly Detection Method=All Methods (Flow Rate, Signal Acquisition, Dynamic Range), Time Feature=Time, Second fraction FR = 0.1, Alpha FR = 0.01, Changepoint Detection Penalty FS = 500, Max Changepoints FS = 3, Dynamic Range Check Side FM=Both, upper and lower limit, Negative Value Removal Method FM = 1. FlowAI-”passed events” were used for further analysis, and all samples were subsampled accordingly. Dimensionality reduction and visualization were performed in OMIQ on 100000 Live (viability dye negative) subsampled events per file via Uniform Manifold Approximation and Projection (UMAP), with the settings as follows: Neighbors=15, Minimum Distance=0.4, Components=2, Metric=Euclidean, Learning Rate=1, Epochs=200, Random Seed=6237. All markers were selected as clustering features aside from the viability dye. Clustered Heatmap was performed in OMIQ on 100000 Live (viability dye negative) subsampled events per file, with the following settings: Measure=Medians, Filter=CD3+CD45+, Cluster Rows, Row Distance=euclidean, Row Linkage=ward, Cluster Columns, Column Distance=euclidean, Column Linkage=ward. All markers were selected as clustering features aside from the viability dye, CD3, and CD45. The exhaustion score of TIL samples and three reference benchmark CD19 CAR-T cell products was determined as described by Weber et al. ( 18 ), but using the integrated MFI (iMFI = % positive cells x MFI) with both, percentage of positive expression and MFI data exported from OMIQ for the markers PD-1, TIM-3, LAG3, CD244, CTLA-4, BTLA and CD39. 2.7 Immunohistochemical analysis and scoring spatial immune phenotypes Immunohistochemical (IHC) analysis for CD3 and CD8 was performed on tumor tissue sections derived from the same resection specimens used for TIL expansion. All IHC staining procedures were conducted using the fully-automated Benchmark ULTRA platform (Roche, Ventana Medical Systems, Oro Valley, AZ, USA) under standardized laboratory conditions accredited following the ISO15189 quality system. In short, paraffin tissue sections (3 µm) were incubated with antibodies against CD3 (R.T.U, Ventana, 2GV6), and CD8 (1:20, Dako, C8/144B). As external control, a suitable tissue section was added to each slide. The tumor invasive margins were evaluated by a pathologist according to previously described spatial immune phenotypes, namely immune-desert, immune-excluded, and immune-inflamed ( 19 , 20 ). 2.8 Statistical analysis Statistical analyses were performed using Graphpad Prism (version 10.2.3 for Windows, GraphPad Software, Boston, Massachusetts USA, www.graphpad.com ). Differences in end-cell counts in the pre-REP phase comparing HS and HPL were tested using the Mann–Whitney test. The paired effect estimate for TIL expansion was calculated as the ratio of the mean difference to the difference in standard deviations using (mean HS - mean HPL)/(SD HS - SD HPL). Differences in phenotypes were tested using the Wilcoxon test for paired samples or the paired t-test. All analyses in the REP phase were performed using the Friedman test followed by Dunn’s post hoc test. A p -value of <0.05 was considered statistically significant. 3 Results 3.1 Patient characteristics EC tissue samples were collected from ten patients who underwent standard-of-care hysterectomy between June and September 2023. Detailed clinicopathological characteristics of the patients are presented in Table 1 . The median age was 67 years (range: 52–79 years). Most tumors were of non-endometrioid histotype (70%). Six out of ten patients had FIGO 2009 stage 1 disease (60%). Tumors with low tumor mutational burden (TMB), e.g. with p53 mutations or classified as the NSMP histotype, were most common (60%). Table 1 Patient characteristics (n=10) Age at enrolment (years) Median (range) 67 (52-79) Histotype (n (%)) Endometrioid 3 (30) Non-endometrioid Serous adenocarcinoma 3 (30) Mixed adenocarcinoma a,b 2 (20) Adenosarcoma 1 (10) Undifferentiated uterine sarcoma 1 (10) Grade (n (%)) Endometrioid Grade 3 3 (30) FIGO stage c (n (%)) I (IA – IC) 6 (60) II (IIA – IIB) 0 (0) III (IIIA – IIIC) 4 (40) Molecular subtype (n (%)) TMB high POLE mutation 1 (10) MMR deficient d 3 (30) TMB low P53 mutant 4 (40) NSMP e,f 2 (20) Characteristics of the patients who underwent hysterectomy and from whom the EC samples were obtained. a EC674 60% clear cell and 40% serous. b EC687 70% endometrioid and 30% clear cell. c The revised 2009 FIGO staging for endometrial cancer was used for staging patients following standard-of-care hysterectomy. d All 3 patients MLH1 and PMS2 deficient. e EC703 ER negative. f EC693 some small areas of slightly positive ER. FIGO, Fédération Internationale de Gynécologie et d’Obstétrique; TMB, tumor mutational burden; POLE, DNA polymerase epsilon; MMR, mismatch repair; and NSMP, no specific molecular profile. 3.2 Expansion and phenotype of HS- and HPL-expanded pre-REP TILs We observed comparable TIL expansion and phenotypic profile in the pre-REP phase when comparing HPL medium and HS medium ( Figures 1a, b ). We were able to expand TILs from 7 out of 10 HS samples (70%) and 6 out of 10 HPL samples (60%), for both TMB-high and TMB-low tumors, even though TMB-low tumors generally exhibit fewer TILs in the tumor microenvironment (TME) ( Figures 1b–g, i ). There were no significant differences between TILs expanded using HS medium and HPL medium ( p = 0.84, Figure 1b ). The mean number of expanded TILs was 43.79 x10 6 cells (SD: 31.1, range: 13-99x10 6 ) in HS medium and 60.03 x10 6 cells (SD: 48.43, range: 12.8-134x10 6 ) in HPL medium, corresponding to a mean-to-standard deviation difference ratio of 0.95. TILs were expanded for a mean of 21 days (range, 15–29 days). There were no significant differences between cell numbers when HPL and HS culture was compared within TMB-high ( p = 0.5) or TMB-low tumors ( p = 0.5) ( Figure 1b ). Figure 1 Pre-rapid expansion and phenotype of TILs from endometrial cancer cultured in HS and HPL medium. (a) Schematic overview of the experimental setup. (b) End cell count of TILs after pre-REP culture in HS medium (grey) or HPL medium (blue) for TMB-high (n=3, left) and TMB-low (n=4, right) histotypes. Data from the same patients are horizontally connected. Statistical significance was assessed using the Mann–Whitney test. (c–g, i) Phen
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