Establishment of a humanized patient-derived xenograft mouse model of high-grade serous ovarian cancer for preclinical evaluation of combination immunotherapy

Availability This study did not generate new unique reagents. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 30 Data and Code Availability All data reported in this paper will be shared by the lead contact upon request. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

We thank the patients for their consent and participation in the study. We thank Brith Bergum and Jørn Skavland at the Flow Cytometry Core Facility of the University of Bergen for providing support for our flow cytometry work, as well as Hege Avsnes Dale and Endy Spriet at the Molecular Imaging Center of the University of Bergen for their assistance with tissue imaging. We also declare the use of the BioRender.com platform for figure creation. This research was funded by the Western Norway Regional Health Authority ( project number 28543) and the Research Council of Norway through its Centers of Excellence funding scheme (project number 223250). CRediT Authorship Contribution Statement Luka Tandaric : Conceptualization, Methodology, Formal Analysis, Investigation, Resources, Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization; Line Bjørge: Conceptualization, Methodology, Investigation, Resources, .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 31 Writing - Original Draft, Writing - Review & Editing, Supervision, Project Administration, Funding Acquisition; Martine Rott Lode: Formal Analysis, Investigation, Data Curation, Writing - Review & Editing; Cecilie Fredvik Torkildsen: Methodology, Investigation, Resources, Writing - Review & Editing; Pia Aehnlich: Methodology, Formal Analysis, Investigation, Resources, Data Curation, Writing - Review & Editing, Visualization; Rammah Elnour: Methodology, Resources, Data Curation, Writing - Review & Editing ; Daniela Elena Costea: Methodology, Formal Analysis, Investigation, Resources, Writing - Review & Editing ; Lars Andreas Akslen: Resources, Writing - Review & Editing, Supervision, Funding Acquisition; Liv Cecilie Vestrheim Thomsen: Writing - Original Draft, Writing - Review & Editing, Supervision; Emmet Mc Cormack: Resources, Writing - Review & Editing, Supervision, Funding Acquisition; Katrin Kleinmanns: Conceptualization, Methodology, Formal Analysis, Investigation, Resources, Data Curation, Writing - Original Draft, Writing - Review & Editing, Visualization, Supervision, Project Administration Declaration of Interests L.B. reports leadership roles in Onkologisk Forum between 2018 and 2022, and in the Nordic Society of Gynaecological Oncology (NSGO) and NSGO - Clinical Trials Unit between 2021 and 2024; receipt of a research grant for a researcher -initiated trial in ovarian cancer from AstraZeneca; and receipt of honoraria for holding lectures from GlaxoSmithKline. L.C.V.T. reports receipt of financial support for a researcher-initiated trial from AstraZeneca; and receipt of p ersonal fees from Bayer, Eisai Co. and AstraZe neca. E.M.C. reports share ownership in, and chairing the board of KinN Therapeutics AS. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 32

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J Immunol. 2011; 6: 3186-3197. https://doi.org/10.4049/jimmunol.1101649 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 37 Fig. 1 Establishment and application of the mouse model. (A) Timeline for the generation, monitoring, and combination immunotherapy treatment of a murine orthotopic PDX model of treatment-naïve high-grade serous ovarian cancer. (B) Distribution of model mice and description of immunotherapy administration across treatment groups. HSC - hematopoietic stem cell; PDX - patient-derived xenograft; G.A. - group assignment; BIW - twice a week; IP - intraperitoneally; CTRL - control group; DUR - durvalumab-only group; OLE - oleclumab-only group; DUR+OLE - combination treatment group .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 38 Fig. 2 Monitoring of tumor burden in PDX -implanted experimental mice. (A) Longitudinal overview of weekly bioluminescence imaging results for PDX -implanted mice in the lateral position. Each column represents an individual mouse. Only images from baseline (week 11) through endpoint (week 17) are shown. Mice outlined with an orange border displayed inexplicably low bioluminescence at the specified timepoint, even after luciferin re-injection. These data were excluded from further analyses. Bioluminescence im ages of the mice taken ventrally are displayed in Fig. S5. Full data on the total lateral flux are available in Table S5. (B) Average total lateral (left graph) and ventral (right graph) photon flux in each treatment group during treatment, relative to baseline (marked by a “B” on the X -axis). (C) Comparison of tumor burden at the end of the study between treatment groups. Only the primary tumor was included in tumor burden assessment due to the small size of the metastatic lesions. Tumor volume data is ava ilable in Table S6. CTRL - control group; DUR - durvalumab-only group; OLE - oleclumab-only group; DUR+OLE - combination treatment group .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 39 Fig. 3 Intratumoral leukocyte densities determined by immunohistochemistry (IHC), and digital analysis using QuPath software. (A) Representative images of tumor areas rich in leukocytes selected for positive cell quantification. The top-left image is a schematic representation of the annotation method: By tracing the tumor- stroma border of the invasive margin with a 400 -µm-thick brush tool, an invasive tumor margin of symmetrical intra-stromal and intra-tumoral depths of 200 µm was delineated. The remaining images display serial primary PDX tumor sections stained with antibodies targeting the leukocyte marker specified in the upper -left corner of each image. Antibody details are provided in Table S2. (B) Comparison of intratumoral leukocyte densities between treatment groups. Full data on intratumoral leukocyte density is available in Table S8. (C) Inter -group comparison of the ratios of CD8 +/cytotoxic (Tc) and FoxP3+/regulatory (Treg) TIL densities. (D) Plots depicting correlations between tumor burden at th e end of the study and the densities of intratumoral marker -positive leukocytes for the group of mice treated with durvalumab. Significant correlations are marked with an asterisk (*) in the graph title. All correlation plots are displayed in Fig. S7, and full correlation data is av ailable in Table S9. CTRL - control group; DUR - durvalumab-only group; OLE - oleclumab-only group; DUR+OLE - combination treatment group; Tc - cytotoxic (CD8+) T cell; Treg - regulatory (FoxP3+) T cell; r - Pearson’s correlation coefficient .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 40 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 41 Fig. 4 Results of the spectral flow cytometry analysis of blood samples taken at the end of the study. Data on the human leukocyte counts are available in Table S10. (A) Comparison of total human leukocyte and T -cell subset frequencies per volume of blood between treatment groups. Data on the human leukocyte counts per µL of blood are available in Table S11. (B) Distribution of T -cell subsets across treatment groups, relative to total human leukocytes. Data on the abundances of human leukocyte subsets relat ive to total human leukocytes in the blood are available in Table S12. CTRL - control group; DUR - durvalumab-only group; OLE - oleclumab-only group; DUR+OLE - combination treatment group .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 42 Fig. S1 CD73 expression profiles of the constituents of the dissociated PDX material used in this study. Gates encompass cells above the CD73 positivity threshold, with the relative abundance of CD73-positive cells within the specified cell population displayed on top of each gate. The single-cell data used for this analysis was previously acquired using suspension mass cytometry. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 43 Fig. S2 Gating strategy used for assess ing the purity of samples enriched with human CD34 + hematopoietic stem cells from umbilical cord blood prior to their intravenous injection into NSGS mice. Enrichment was performed by magnetic activated cell sorting. Samples were analyzed using conventional flow cytometry. SSC - side scatter; FSC - forward scatter; A - area; H - height; HSC - hematopoietic stem cell; MACS - magnetic activated cell sorting .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 44 Fig. S3 Representative gating strategy for the assessment of blood chimerism in mice injected with human hematopoietic stem cells. Leukocyte phenotyping was performed using fluorescence flow cytometry. Complete blood chimerism data is available in Table S4. SSC - side scatter; FSC - forward scatter; A - area; H - height; mCD45 - murine CD45; hCD45 - human CD45 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 45 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 46 Fig. S4 Key elements of the workflow used for the analysis of endpoint blood samples by spectral flow cytometry. (A) Titration of the anti -PD-L1 antibody. The sample used for titration consisted of peripheral blood mononuclear cells stimulated with phytohemagglutinin. The remainder of the spectral flow cytometry panel was titrated prior to this study (unpublished data). (B) Representative gating strategy used for the characterization of blood sample composition. (C) Histograms representing PD-L1 expression profiles of the total T-cell populations relative to the unstained control. Mouse IDs are displayed to the right of each histogram. Certain samples were excluded from this analysis due to low T-cell counts. Full PD-L1 expression data is available in Table S13. SSC - side scatter; FSC - forward scatter; Tn - naïve T cells; Tcm - central memory T cells; Tem - effector memory T cells; Temra - effector T cells; CTRL - control group; DUR - durvalumab-only group; OLE - oleclumab-only group; DUR+OLE - combination treatment group .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 47 Fig. S5 Longitudinal overview of weekly bioluminescence imaging results for PDX -implanted experimental mice in the ventral position. Each column represents an individual mouse. Only images from baseline (week 11) through endpoint (week 17) are shown. Mice outline d with an orange border displayed inexplicably low bioluminescence at the specified timepoint, even after luciferin re -injection. These data were excluded from further analyses. Full data on the total ventral flux are available in Table S5. CTRL - control group; DUR - durvalumab-only group; OLE - oleclumab-only group; DUR+OLE - combination treatment group .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 48 Fig. S6 Light microscopy images (400x) of representative areas of the primary PDX tumor displaying prominent accumulation of human leukocytes in the invasive margin. Tumor sections were stained for human CD45. Each image encompasses a 2.5 mm 2 area of a representative PDX tumor section from each treatment group (specified in the top left corner of each image). CTRL - control group; DUR - durvalumab-only group; OLE - oleclumab-only group; DUR+OLE - combination treatment group; PDX - patient-derived xenograft .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 49 Fig. S7 Correlation plots showing associations between tumor burden at the end of the study and densities of intratumoral marker-positive leukocytes. Top row: all samples combined. Bottom four rows: samples from individual treatment groups. Significant correlation s (p<0.05) are marked with an asterisk (*) in the graph title. Full correlation data is available in Table S9. CTRL - control group; DUR - durvalumab-only group; OLE - oleclumab-only group; DUR+OLE - combination treatment group .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 50 Table S1 Antibody panel used for the characterization of leukocytes in the blood samples from the experimental mice using spectral flow cytometry. Fluorophore Target Clone Dilution Vendor Cat.No. BUV615 CD3 HIT3a 1:160 BD 751157 PerCP/Fire 806 CD4 SK3 1:160 BioLegend 344694 Spark Blue 550 CD8 SK1 1:160 BioLegend 344760 PE/Fire 640 CD19 HIB19 1:160 BioLegend 302274 Spark Red 718 CD27 QA17A18 1:80 BioLegend 393218 BV480 CD45RO UCHL1 1:160 BD 566143 BUV 805 CD45 HI30 1:160 ThermoFisher 368-0459-42 BV711 PD-L1 29E.2A3 1:80 BioLegend 329722 BD - Becton, Dickinson and Company; BUV - Brilliant Ultra Violet; BV - Brilliant Violet .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 51 Table S2 List of antibodies used for the immunohistochemical staining of primary patient-derived xenograft tumor sections. Target Clone Host Species Dilution Vendor Cat.No. hCD45 2B11 Mouse 1:100 ThermoFisher 14-9457-82 CD20 H1 Mouse 1:200 BD 555677 CD3 F7.2.38 Mouse 1:200 Abcam Ab17143 CD8 C8/144B Mouse 1:400 BioLegend 372902 FoxP3 236A/E7 Mouse 1:50 ThermoFisher 14-4777-82 BD - Becton, Dickinson and Company; hCD45 - Human CD45 .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 52 Table S3 Overview of the positive cell detection parameters used for the enumeration of leukocytes in primary PDX tumor sections. Parameter hCD45, CD3, CD8 CD20 FoxP3 Detection image Optical density sum Requested pixel size 0.17 µm

Radius 8 µm Use opening by reconstruction Yes Median filter radius 0 Sigma 1.5 Minimum area 20 Maximum area 201 Threshold 0.1 Max background intensity 2 Split by shape Yes Exclude DAB (membrane staining) No Cell expansion 3 µm Include cell nucleus Yes Smooth boundaries No Make measurements Yes Score compartment Cell: DAB OD Mean Nucleus: DAB OD Mean Nucleus: DAB OD Mean Threshold 1+ 0.04 0.2 0.3 Threshold 2+ 0.4 Threshold 3+ 0.6 Single threshold Yes Yes Yes .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 53 Table S4 Results of the chimerism assessment of mouse blood during model establishment and at the end of the study. Mouse ID Week 8 (PDX Implantation) Week 11 (Group Assignment) Week 17 (End of Study) Humana B Cellsb T Cellsb Humana B Cellsb T Cellsb Humana B Cellsb T Cellsb 01 18.4 % 90.7 % N/A 19.0 % 88.8 % 1.43 % 21.9 % 64.3 % 28.8 % 02 18.4 % 88.5 % N/A 22.6 % 85.3 % 1.96 % 51.6 % 39.7 % 51.3 % 03 19.4 % 89.1 % N/A 19.3 % 88.4 % 1.11 % 24.0 % 91.0 % 0.75 % 04 17.5 % 86.6 % N/A 21.1 % 86.4 % 1.21 % 28.2 % 90.5 % 0.96 % 05 30.9 % 87.4 % N/A 37.0 % 88.8 % 0.75 % 22.6 % 47.9 % 42.7 % 06 18.4 % 86.6 % N/A 19.2 % 82.4 % 2.35 % 29.1 % 56.0 % 31.7 % 07 16.9 % 86.5 % N/A 24.3 % 86.9 % 1.66 % 20.0 % 41.8 % 48.1 % 08 17.7 % 87.0 % N/A 18.6 % 56.2 % 30.7 % 22.2 % 10.7 % 77.3 % 09 14.2 % 87.1 % N/A 13.3 % 86.8 % 1.61 % 11.9 % 78.1 % 14.9 % 10 13.4 % 85.6 % N/A 26.3 % 88.3 % 1.17 % 28.7 % 44.5 % 41.6 % 11 14.1 % 90.8 % N/A 23.0 % 88.8 % 1.10 % 29.4 % 32.2 % 57.4 % 12 14.0 % 88.2 % N/A 17.9 % 87.0 % 1.52 % 25.8 % 88.7 % 0.68 % 13 27.6 % 88.2 % N/A 28.2 % 87.0 % 0.91 % 29.8 % 87.9 % 1.52 % 14 12.1 % 75.0 % N/A 28.8 % 89.6 % 1.30 % 39.4 % 12.2 % 79.6 % 15 26.6 % 88.9 % N/A 31.8 % 87.5 % 1.29 % 20.0 % 75.3 % 2.12 % 16 13.1 % 92.6 % N/A 10.6 % 90.9 % 0.90 % 8.14 % 87.6 % 1.42 % 17 20.9 % 90.4 % N/A 19.8 % 80.3 % 6.66 % 30.4 % 22.9 % 70.6 % 18 13.3 % 88.6 % N/A 18.2 % 86.8 % 1.16 % 14.2 % 82.6 % 9.52 % a Percentage of single cells that are positive for human CD45. b Relative to total cells positive for human CD45. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 54 Table S5 Total lateral and ventral photon flux measured weekly during weeks 9 through 17 after injection of hematopoietic cells into the experimental mice. Mouse ID W09 W10 W11 W12 W13 W14 W15 W16 W17 Total Flux - Lateral 01 1.49E+07 6.66E+07 2.67E+08 1.74E+09 2.43E+09 3.25E+09 5.00E+07 7.69E+09 5.98E+09 02 1.03E+07 2.74E+07 3.86E+08 7.00E+08 1.42E+09 3.38E+09 1.90E+06 3.49E+09 1.11E+10 03 3.93E+05 7.23E+07 2.67E+07 1.22E+09 2.87E+09 4.27E+09 3.76E+06 1.00E+10 2.44E+10 04 4.40E+05 4.84E+07 4.25E+08 1.32E+09 1.72E+06 5.85E+09 6.78E+09 1.11E+10 1.24E+10 05 4.39E+06 1.52E+07 8.25E+07 2.02E+08 1.40E+09 2.19E+09 3.30E+09 5.18E+09 6.60E+09 06 2.43E+07 9.86E+07 7.16E+08 2.92E+06 8.37E+09 6.81E+09 6.05E+09 1.29E+10 2.26E+10 07 4.43E+05 3.69E+07 2.36E+08 5.47E+08 1.48E+09 2.73E+09 4.21E+07 8.16E+09 1.45E+10 08 2.32E+06 8.93E+06 1.00E+08 8.16E+07 2.19E+08 1.53E+09 4.48E+08 3.84E+09 4.88E+09 09 7.80E+05 8.69E+05 2.30E+06 1.72E+06 3.82E+05 2.87E+07 5.17E+07 1.59E+06 2.95E+06 10 1.37E+06 6.96E+07 2.98E+08 2.93E+06 5.05E+09 5.81E+09 1.11E+08 1.10E+10 1.93E+10 11 1.36E+07 7.39E+07 2.35E+08 1.13E+09 2.65E+09 2.37E+09 4.00E+09 1.25E+10 2.44E+10 12 6.18E+05 1.63E+08 6.25E+08 2.42E+09 6.67E+09 1.36E+10 1.17E+10 1.56E+10 4.46E+10 13 4.43E+06 1.63E+07 4.49E+07 2.29E+08 5.03E+08 1.71E+09 2.92E+09 5.52E+09 1.26E+07 14 2.38E+07 6.75E+07 4.28E+08 2.03E+09 4.49E+09 5.52E+09 2.80E+07 1.43E+10 1.53E+07 15 7.69E+06 5.18E+07 3.39E+08 1.01E+09 2.69E+09 4.01E+09 5.42E+09 7.79E+09 7.86E+09 16 1.23E+06 6.33E+06 4.67E+07 7.20E+07 7.39E+08 7.30E+08 1.33E+09 5.80E+08 1.70E+09 17 5.89E+05 1.87E+07 2.22E+08 3.53E+08 6.41E+05 2.26E+07 5.51E+08 1.08E+10 2.11E+10 18 1.00E+06 1.68E+06 1.43E+07 4.19E+07 9.87E+05 2.22E+08 1.27E+09 1.90E+09 5.15E+09 Total Flux - Ventral 01 2.01E+09 2.30E+09 1.13E+11 4.29E+10 8.02E+10 1.07E+11 5.55E+10 8.12E+11 2.69E+12 02 9.34E+08 7.25E+08 1.86E+09 1.70E+10 6.68E+10 1.39E+11 1.07E+09 1.79E+12 1.02E+13 03 4.78E+08 1.13E+09 1.31E+09 2.58E+10 2.13E+11 8.28E+11 2.02E+09 1.01E+13 6.11E+13 04 4.71E+08 2.74E+09 2.96E+09 3.30E+11 6.16E+09 2.27E+11 8.80E+11 2.97E+12 6.97E+12 05 8.04E+08 1.23E+09 3.48E+09 4.16E+09 6.82E+10 4.03E+11 1.49E+12 3.75E+12 1.26E+13 06 1.14E+09 1.41E+09 1.07E+10 8.71E+08 5.27E+11 1.42E+12 2.68E+12 6.89E+12 3.04E+13 07 5.22E+08 1.03E+10 4.50E+10 3.17E+11 2.26E+11 6.15E+11 1.91E+10 4.84E+12 2.43E+13 08 9.86E+08 1.28E+09 2.32E+09 9.14E+06 1.05E+10 2.06E+11 5.00E+11 3.31E+12 7.14E+12 09 1.08E+09 1.07E+09 1.15E+09 6.89E+08 1.00E+11 1.80E+09 2.96E+09 5.99E+08 6.62E+08 10 6.93E+08 1.65E+09 3.16E+09 5.98E+08 1.67E+11 2.41E+11 3.56E+10 4.99E+12 2.67E+13 11 1.34E+09 2.28E+09 8.80E+10 4.50E+10 2.44E+11 5.81E+11 2.63E+12 7.91E+12 4.05E+13 12 5.85E+08 6.96E+09 2.80E+10 2.35E+11 2.83E+11 6.34E+11 2.73E+12 7.68E+12 5.69E+13 13 1.49E+09 1.59E+09 2.02E+09 6.24E+09 3.38E+09 4.48E+10 2.60E+10 6.71E+11 1.27E+09 14 9.20E+08 2.17E+09 1.31E+10 5.20E+10 3.13E+11 7.87E+11 2.04E+11 9.18E+12 6.15E+09 15 1.54E+09 5.09E+09 1.90E+10 8.57E+10 2.88E+11 4.92E+11 2.63E+12 6.89E+12 8.13E+12 16 1.07E+09 8.63E+08 1.86E+09 6.00E+09 2.66E+10 1.57E+10 7.75E+10 6.85E+10 3.30E+11 17 6.70E+08 2.98E+09 1.81E+09 9.92E+09 3.73E+08 5.62E+09 1.94E+11 4.68E+12 3.19E+13 18 1.04E+09 9.25E+08 1.65E+09 1.44E+09 5.75E+08 1.87E+10 1.57E+11 1.15E+12 4.52E+12 W - week .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 55 Table S6 Dimensions of primary patient-derived xenograft tumors measured at the end of the study. Tumor volumes were calculated using the formula: (height x width x length x π) / 6. Mouse ID Tumor Dimensions Height [mm] Width [mm] Length [mm] Volume [mm3] 01 2.8 3.4 5.32 26.52 02 8.35 3.32 13.24 192.18 03 5.73 8.38 9.65 242.62 04 6.47 6.1 7.88 162.84 05 11.27 7.45 10.73 471.71 06 4.99 6.14 10.61 170.21 07 4.34 3.26 6.72 49.78 08 5.8 9.39 8.43 240.39 09 4.2 5.04 6.57 72.82 10 6.92 5.51 4.07 81.26 11 5.03 6.54 5.33 91.81 12 5.43 7.66 6.88 149.84 13 5.17 4.86 3.59 47.23 14 7.4 4.91 8.04 152.96 15 10.09 10.72 14.11 799.12 16 10.75 7.02 7.13 281.73 17 6.2 10.8 5.95 208.61 18a - - - - a Primary tumor too small to measure. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 56 Table S7 The extent of visible metastatic dissemination at the end of the study. Mouse ID Anatomic Localization of Visible Metastatic Lesion(s) DIA DUO GAS HEP MES OME PER REN SPL 01 x x x 02 x x 03 x x 04 x x 05 x x x 06 x x 07 x x x x 08 x x x x 09 10 x x 11 x 12 x x x x 13 x 14 x x x 15 x x x x x x 16 x 17 18 x DIA - diaphragmatic; DUO - duodenal; GAS - gastric; HEP - hepatic; MES - mesenteric; OME - omental; PER - peritoneal wall; REN - renal; SPL - splenic .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 57 Table S8 Results of the digital analysis of primary patient-derived xenograft tumor sections. The total size of the annotated area is expressed as mm2, and the positive cell density is expressed as the total number of positive cells per mm2 of the total annotated area. Mouse ID Parameter Marker hCD45 CD20 CD3 CD8 FoxP3 01 Area 0.65 0.65 0.65 0.51 0.65 Count 226 97 171 16 15 Density 346.4 148.7 262.1 31.1 23.2 02 Area 1.99 1.99 1.97 1.99 1.95 Count 1094 424 1037 324 104 Density 548.5 212.6 526.1 162.4 53.3 03 Area 0.81 0.80 0.81 0.76 0.78 Count 58 30 15 13 5 Density 71.2 37.6 18.4 17.1 6.4 04 Area 1.60 1.60 1.60 1.50 1.60 Count 215 221 6 10 3 Density 134.4 138.2 3.8 6.7 1.9 05 Area 1.47 1.46 1.47 1.47 1.47 Count 459 230 338 141 136 Density 311.3 157.5 229.2 95.6 92.7 06 Area 1.38 1.38 1.37 1.38 1.37 Count 316 74 164 56 9 Density 228.8 53.8 119.6 40.5 6.5 07a - - - - - - 08 Area 0.79 0.79 0.79 0.76 0.79 Count 116 19 141 56 19 Density 146.0 23.9 177.5 73.4 23.9 09 Area 0.90 0.90 0.80 0.90 0.90 Count 37 10 43 22 6 Density 40.9 11.1 53.4 24.3 6.7 10 Area 0.91 0.93 0.93 0.93 0.93 Count 110 35 79 18 14 Density 121.1 37.5 84.6 19.3 15.0 11 Area 1.34 1.29 1.33 1.34 1.34 Count 330 41 174 30 10 Density 247.0 31.8 130.6 22.5 7.5 12 Area 1.00 1.00 1.00 0.99 0.98 Count 68 47 9 15 4 Density 67.8 46.8 9.0 15.1 4.1 13 Area 0.86 0.86 0.85 0.86 0.86 Count 66 54 14 15 2 Density 76.7 62.8 16.5 17.4 2.3 14 Area 0.69 0.69 0.69 0.69 0.69 Count 362 34 341 83 50 Density 521.6 49.0 491.3 119.6 72.0 15 Area 1.15 1.15 0.76 1.06 1.06 Count 782 399 7 12 1 Density 679.0 346.5 9.2 11.3 0.9 16 Area 0.72 0.72 0.72 0.72 0.72 Count 174 238 1 3 22 Density 241.9 330.8 1.4 4.2 30.6 17 Area 2.53 2.52 2.53 2.53 2.43 Count 1034 69 795 411 51 Density 409.4 27.4 314.8 162.7 21.0 18 Area 0.86 0.82 0.86 0.83 0.86 Count 34 22 38 49 9 Density 39.4 26.7 44.0 59.3 10.4 a No discernable invasive tumor margin present. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 58 Table S9 Parameters of the correlations between tumor burden at the end of the study and densities of intratumoral marker-positive leukocytes for all samples combined and each treatment group individually. Treatment Group Parameter Pearson’s Correlation Coefficient (r) Goodness of Fit (R2) p-value hCD45 CD20 CD3 CD8 FoxP3 All Samples ra 0.382 0.359 -0.082 -0.029 0.159 R2 0.317 0.433 0.016 0.001 0.029 p 0.145 0.173 0.763 0.917 0.556 Control r -0.299 -0.368 -0.195 0.167 -0.079 R2 0.089 0.135 0.038 0.028 0.006 p 0.701 0.632 0.805 0.833 0.921 Durvalumab r 0.853 0.922 0.950 0.952 0.961 R2 0.728 0.850 0.902 0.907 0.924 p 0.147 0.078 0.050 0.048 0.039 Oleclumab r -0.107 -0.351 -0.173 -0.392 -0.061 R2 0.012 0.123 0.030 0.153 0.004 p 0.893 0.649 0.828 0.608 0.939 Durvalumab + Oleclumab r 0.659 0.720 -0.680 -0.638 -0.756 R2 0.434 0.518 0.463 0.407 0.571 p 0.341 0.280 0.320 0.362 0.244 r - Pearson’s correlation coefficient R2 - goodness of fit parameter of the results of simple linear regression p - p-value a Spearman’s rank correlation used due to non-normality of dataset. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 59 Table S10 Results of the spectral flow cytometry analysis of leukocytes in the blood samples from the experimental mice - human leukocyte counts. Mouse ID Total Human Leukocytes T Cells Total CD4+ CD8+ Total N CM EM E Total N CM EM E 01 11758 2687 2281 115 1976 188 2 317 80 237 0 0 02 92523 47690 42615 997 29170 12211 237 4197 472 3720 5 0 03 20376 24 2 1 0 1 0 8 7 1 0 0 04 11458 18 0 0 0 0 0 1 1 0 0 0 05 15147 6823 5057 369 4472 207 9 1333 327 1000 5 1 06 27069 7650 4705 214 3300 1169 22 1583 154 1400 25 4 07 4735 2028 1948 95 1519 330 4 50 9 40 0 1 08 4726 3856 3344 30 2731 571 12 313 36 268 7 2 09 4616 345 257 67 121 65 4 48 27 21 0 0 10 14045 5025 4216 113 3035 1057 11 453 44 408 1 0 11 23690 14465 12695 252 10262 2157 24 1281 207 1069 3 2 12 25128 28 0 0 0 0 0 3 3 0 0 0 13 23542 109 48 15 30 3 0 32 26 4 0 2 14 16152 13406 11552 334 8703 2391 124 731 82 649 0 0 15 11661 28 14 4 10 0 0 6 5 1 0 0 16 9711 2 0 0 0 0 0 0 0 0 0 0 17 47863 35237 26539 1130 22368 2821 220 6140 297 5805 27 11 18 15658 1145 654 63 332 245 14 249 141 105 2 1 CM - Central Memory; E - Effector (Temra); EM - Effector Memory; N - Naïve .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 60 Table S11 Results of the spectral flow cytometry analysis of leukocytes in the blood samples from the experimental mice - human leukocyte counts per µL of blood. Mouse ID Total Human Leukocytes T Cells Total CD4+ CD8+ Total N CM EM E Total N CM EM E 01 91.6 20.9 17.8 0.9 15.4 1.5 0.0 2.5 0.6 1.8 0.0 0.0 02 608.6 313.7 280.3 6.6 191.9 80.3 1.6 27.6 3.1 24.5 0.0 0.0 03 168.3 0.2 0.0 0.0 0.0 0.0 0.0 0.1 0.1 0.0 0.0 0.0 04 186.8 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 05 140.5 63.3 46.9 3.4 41.5 1.9 0.1 12.4 3.0 9.3 0.0 0.0 06 377.2 106.6 65.6 3.0 46.0 16.3 0.3 22.1 2.1 19.5 0.3 0.1 07 38.7 16.6 15.9 0.8 12.4 2.7 0.0 0.4 0.1 0.3 0.0 0.0 08 73.5 59.9 52.0 0.5 42.5 8.9 0.2 4.9 0.6 4.2 0.1 0.0 09 56.5 4.2 3.1 0.8 1.5 0.8 0.0 0.6 0.3 0.3 0.0 0.0 10 203.0 72.6 60.9 1.6 43.9 15.3 0.2 6.5 0.6 5.9 0.0 0.0 11 369.9 225.9 198.2 3.9 160.2 33.7 0.4 20.0 3.2 16.7 0.0 0.0 12 228.6 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 13 185.9 0.9 0.4 0.1 0.2 0.0 0.0 0.3 0.2 0.0 0.0 0.0 14 157.6 130.8 112.7 3.3 84.9 23.3 1.2 7.1 0.8 6.3 0.0 0.0 15 215.3 0.5 0.3 0.1 0.2 0.0 0.0 0.1 0.1 0.0 0.0 0.0 16 93.5 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 17 420.9 309.8 233.4 9.9 196.7 24.8 1.9 54.0 2.6 51.0 0.2 0.1 18 107.1 7.8 4.5 0.4 2.3 1.7 0.1 1.7 1.0 0.7 0.0 0.0 CM - Central Memory; E - Effector (Temra); EM - Effector Memory; N - Naive .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 61 Table S12 Results of the spectral flow cytometry analysis of leukocytes in the blood samples from the experimental mice - human leukocyte abundances relative to total human leukocytes. Values in the cells are expressed as percentages. Mouse ID Total Human Leukocytes T Cells Total CD4+ CD8+ Total N CM EM E Total N CM EM E 01 100.0 22.9 19.4 1.0 16.8 1.6 0.0 2.7 0.7 2.0 0.0 0.0 02 100.0 51.5 46.1 1.1 31.5 13.2 0.3 4.5 0.5 4.0 0.0 0.0 03 100.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 04 100.0 0.2 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 05 100.0 45.0 33.4 2.4 29.5 1.4 0.1 8.8 2.2 6.6 0.0 0.0 06 100.0 28.3 17.4 0.8 12.2 4.3 0.1 5.8 0.6 5.2 0.1 0.0 07 100.0 42.8 41.1 2.0 32.1 7.0 0.1 1.1 0.2 0.8 0.0 0.0 08 100.0 81.6 70.8 0.6 57.8 12.1 0.3 6.6 0.8 5.7 0.1 0.0 09 100.0 7.5 5.6 1.5 2.6 1.4 0.1 1.0 0.6 0.5 0.0 0.0 10 100.0 35.8 30.0 0.8 21.6 7.5 0.1 3.2 0.3 2.9 0.0 0.0 11 100.0 61.1 53.6 1.1 43.3 9.1 0.1 5.4 0.9 4.5 0.0 0.0 12 100.0 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 13 100.0 0.5 0.2 0.1 0.1 0.0 0.0 0.1 0.1 0.0 0.0 0.0 14 100.0 83.0 71.5 2.1 53.9 14.8 0.8 4.5 0.5 4.0 0.0 0.0 15 100.0 0.2 0.1 0.0 0.1 0.0 0.0 0.1 0.0 0.0 0.0 0.0 16 100.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 17 100.0 73.6 55.4 2.4 46.7 5.9 0.5 12.8 0.6 12.1 0.1 0.0 18 100.0 7.3 4.2 0.4 2.1 1.6 0.1 1.6 0.9 0.7 0.0 0.0 CM - Central Memory; E - Effector (Temra); EM - Effector Memory; N - Naïve .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 62 Table S13 PD-L1 expression levels on T cells and relative abundances of PD-L1-positive T cells in mouse blood taken at the end of the study. Mouse ID PD-L1 MFI T Cellsa PD-L1 MFI CD4+ Cellsa %PD-L1+ CD4+ Cellsb PD-L1 MFI CD8+ Cellsa %PD-L1+ CD8+ Cellsb 01 118 117 0,088 146 0,32 02 35,8 33,1 0,12 53,7 0,17 03c 19,7 -70,8 0 -7,15 0 04c -427 N/A 0 -267 0 05 125 142 0,26 80,7 0,15 06 -369 -352 0,28 -417 0,13 07 102 98,8 0,15 189 0 08 -436 -437 0,06 -456 0 09 -382 -350 0 -441 0 10 23,2 16,1 0,17 78,9 0,44 11 -368 -370 0,047 -366 0,078 12c 54,6 N/A 0 -53,7 0 13 71,7 140 0 -29,5 0 14 15,2 8,94 0,061 115 0,14 15c -287 -263 0 -799 0 16c 183 N/A 0 N/A 0 17 19,7 20,6 0,064 8,04 0,016 18 78 108 0,46 29,5 0 MFI - median fluorescence intensity a Expressed as median fluorescence intensity relative to an unstained control sample. b Abundance is relative to total CD4+ or CD8+ T-cell population. c Sample contained insufficient T-cells for measuring PD-L1 expression. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 63 Supplementary Protocol: Isolation of Hematopoietic Stem Cells from Umbilical Cord Blood Ensure that the working environment and solutions used in this procedure are clean and sterile. Solutions should be acclimated to room temperature (RT), unless specified otherwise in the protocol. While the choice of density gradient medium is left up to the reader, this protocol is based on the usage of Lymphoprep™ (Cat.No. 07861, Stemcell Technologies, Canada). Lymphoprep™ should be protected from long exposure to light. 1 – Isolation of Mononuclear Cells (MNCs) by Density Gradient Centrifugation 1) Determine the total volume of the umbilical cord blood. 2) Aliquot a volume of density gradient medium equal to the volume of the umbilical cord blood into 50 mL conical centrifugation tubes (henceforth referred to as “50 mL tubes”). The maximum volume of density gradient medium in each 50 mL tube should not exceed 15 mL. 3) Dilute the umbilical cord blood using an equal volume of phosphate-buffered saline (PBS) or a 0.9% w/V solution of NaCl (saline). 4) Gently dispense the diluted umbilical cord blood onto the top of the density gradient medium. The volume of added blood should equal to double the volume of the density gradient medium. 5) Centrifuge the samples (400 g, 30 min, RT, with acceleration set to minimum and brakes disabled). 6) Transfer the MNCs forming the cloudy layer between the plasma and the density gradient medium into new 50 mL tubes. Ensure the maximum possible recovery of MNCs. Avoid pooling MNCs from different tubes. 7) Resuspend the MNCs in each 50 mL tube to 45 mL of total volume by adding PBS/saline and gently inverting the tubes multiple times. 8) Centrifuge the MNC suspensions (500 g, 5 min, RT). 9) Remove the supernatant by aspiration. Avoid decanting. 10) Resuspend the pelleted MNCs by adding 2 mL of PBS/saline and gently vortexing. 11) Add 20 mL of 1x Red Blood Cell Lysis Buffer (Cat.No. TNB-4300, Cytek Biosciences, USA) to each MNC suspension. 12) Gently invert the tubes multiple times to mix. 13) Incubate the MNCs in 1x Red Blood Cell Lysis Buffer (8 min, RT, protected from light). 14) Add magnetic-activated cell sorting (MACS) buffer (solution of 2 mM EDTA and 0.5% w/V bovine serum albumin in PBS, pH 7.2) into the MNC suspensions for a total suspension volume of 45 mL. 15) Gently invert the tubes multiple times to mix. 16) Centrifuge the MNC suspensions (300 g, 10 min, RT). 17) Remove the supernatant by aspiration. Avoid decanting. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 64 * The MNCs can be frozen at this step for purification of CD34+ cells at a later timepoint. Once thawed, pooled and centrifuged, continue with the protocol from step 19. 18) If there are multiple tubes containing MNCs, resuspend each pellet in 1 mL of MACS buffer by pipetting and pool all of the MNC suspensions into a new 50 mL tube. * From this point in the protocol onwards, work with cold solutions (4°C) and keep cells in a cold environment, unless specified otherwise. 19) Resuspend the MNCs in 40 mL of MACS buffer. 20) Filter the MNC suspension through a cell strainer with a pore size of 30-40 µm into a new 50 mL tube. 21) Determine the cell count. 22) Centrifuge the MNC suspension (300 g, 10 min, 4°C). 23) Remove the supernatant by aspiration. Avoid decanting. 2 – Purification of CD34+ Human Hematopoietic Stem Cells 24) Resuspend the MNCs in MACS buffer. The total volume of the MNC suspension should be 300 µL if there are 1 x 108 MNCs or fewer. For higher cell counts, scale the suspension volume proportionally so there are 1 x 10 8 MNCs per 300 µL of suspension. During the purification of hematopoietic stem cells, scale the volumes of used reagents in the same manner. * The following CD34 + cell purification protocol has been adapted from that of the CD34 MicroBead Kit (human) (Cat. No. 130-046-703, Miltenyi Biotec, Germany). 25) For every 1 x 108 cells, add 100 µL of human FcR blocking reagent into the MNC suspension. 26) For every 1 x 108 cells, add 100 µL of CD34 microbeads into the MNC suspension. 27) Mix the MNC suspension by vortexing. 28) Incubate the MNC suspension on ice for 30 minutes, gently vortexing the suspension every 10 minutes. 29) For every 1 x 108 cells, add 5 mL of MACS buffer to the MNC suspension. 30) Centrifuge the MNC suspension (300 g, 10 min, 4°C). 31) Remove as much of the supernatant as possible by aspiration. Avoid decanting. 32) For every 1 x 108 cells, add 500 µL of MACS buffer to the MNC pellet. 33) Resuspend the pelleted MNCs by gently vortexing. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 65 34) Move an aliquot of the MNC suspension containing 2 x 10 6 cells into a separate tube for later flow cytometric analysis. Keep the aliquoted “pre-MACS” sample of MNCs cold. 35) Place an LS column into a MACS separator. 36) Rinse the LS column with 3 mL of MACS buffer and discard the flow-through. 37) Apply the MNC suspension onto the LS column and collect the flow-through fraction. 38) Wash the LS column with three 3 mL portions of MACS buffer and collect the flow -through fraction into the same tube as in step 37. 39) Move the LS column out of the magnetic field of the MACS separator and place it onto a 15 mL conical centrifugation tube. 40) Add 5 mL of MACS buffer onto the column and immediately force the added liquid through the LS column using the LS column’s plunger, collecting the eluate (the hematopoietic stem cell (HSC)-enriched fraction) into the 15 mL tube. 41) Perform steps 35-40 on the HSC-enriched fraction using a second LS column. Use the tube used in step 37 for the collection of unenriched flow-through fraction. 42) Determine the cell count in the flow -through fraction and the count and viability of the cells in the HSC - enriched fraction. 43) Move an aliquot of 1-2 x 106 cells from the flow-through fraction and an aliquot of 1.5-2.5 x 104 cells from the HSC-enriched fraction into separate tubes for flow cytometric analysis. 44) Keep the HSC-enriched fraction cold while determining HSC purity in cell aliquots via flow cytometry. 3 – Determination of the Purity of Isolated CD34+ Human Hematopoietic Stem Cells 45) Centrifuge the aliquots of pre-MACS, flow-through and HSC-enriched cells (450 g, 5 min, RT). 46) Remove as much supernatant as possible while minimizing cell loss. 47) Resuspend the pelleted cell aliquots. For every 5 x 106 cells, rounded up, add 50 µL of PBS to the pellet. 48) Separate out an aliquot from the pre-MACS cell sample for use as an unstained control. 49) Stain the cell aliquots using an anti -human CD34 antibody (Cat.No. 130 -098-140, Miltenyi Biotec, Germany). 50) Incubate antibody-stained cells for 10 minutes in a refrigerator (4°C). 51) Centrifuge the stained cell aliquots (450 g, 5 min, 4°C). 52) Aspirate supernatant. 53) Resuspend the cells in 250 µL of MACS buffer. 54) Acquire data on a flow cytometer. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 66 Supplementary Protocol: Preparation of Blood Samples for Spectral Flow Cytometry Analysis 1 – Thawing and Washing 1) Thaw the cryovials containing the mouse blood samples fixed using Stable-Lyse2 (Cat.No STBLYSE2- 250, Smart Tube, USA) and Stable-Store2 (Cat.No STBLSTORE2-1000, Smart Tube, USA) at 4°C. 2) While the samples are thawing: a. Label one 5 mL round-bottom tube for each sample. b. Prepare 2 mL of 0,25 mg/mL DNAse I (Cat.No. DN25, Sigma-Aldrich, USA) in Dulbecco’s phosphate-buffered saline containing Ca2+ and Mg2+ (Cat.No. D8662, Sigma-Aldrich, USA) per sample and aliquot 1 mL of the DNAse solution into each labeled 5 mL tube. Acclimate the DNAse solution to room temperature (RT). c. Acclimate CountBright Absolute Counting Beads (Cat.No. C36950, ThermoFisher Scientific, USA) to RT. Vortex the beads thoroughly. Into each labeled 5 mL tube containing DNAse solution, add 1 x 104 counting beads for every 50 µL of blood (320 µL of fixed blood) constituting that sample. 3) One cryovial at a time: a. Pipet the contents of the cryovial gently and thoroughly to resuspend the cells. b. Transfer the contents of the cryovial into the corresponding 5 mL tube containing DNAse solution and counting beads. c. Add 1 mL of DNAse solution into the cryovial. d. Wash out the cryovial with the DNAse solution and transfer the washout to the corresponding 5 mL tube. e. Pipet gently and thoroughly to mix the contents of the 5 mL tube. 4) Incubate cells in DNAse solution for a minimum of 10 minutes. 5) Centrifuge the samples (800 g, 5 min, RT). 6) Remove supernatant by pipetting. 7) Add 1 mL of phosphate-buffered saline (PBS) to each sample. 8) Resuspend the cells by vortexing. 9) One sample at a time: a. Pipet the resuspended cells through the filter of a correspondingly labeled filter-capped 5 mL tube (Cat.No. 352235, Corning, USA). b. Add 1 mL of PBS to the original 5 mL tube. c. Wash the walls of the tube by vortexing. d. Transfer the washout through the filter of the corresponding 5 mL tube. 10) Centrifuge the samples (800 g, 5 min, RT). 11) Remove as much supernatant as possible by pipetting. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 67 2 – Staining 12) Prepare FcR blocking buffer: a. 38 µL of base buffer (2% V/V fetal bovine serum in PBS) per sample. b. 10 µL of human FcR blocking agent (Cat.No. 130-059-901, Miltenyi Biotec, Germany) per sample. c. 2 µL of anti-mouse-CD16/CD32 monoclonal antibody (Cat.No. 16-0161-82, ThermoFisher Scientific, USA) per sample. 13) Add 50 µL of FcR blocking buffer to each cell pellet. 14) Resuspend the cells by gently vortexing. 15) Incubate cells in FcR blocking buffer for 10 minutes at RT. 16) Add 50 µL of antibody mix to each sample. 17) Mix by gently vortexing. 18) Incubate cells in the antibody mix for 30 minutes in a fridge (4°C). 19) Add 2 mL of base buffer to each sample. 20) Mix by vortexing. 21) Centrifuge the samples (800 g, 5 min, RT). 22) Remove supernatant by pipetting. 23) Repeat steps 19-22 to perform a second cell wash. 24) Resuspend the cells by gently vortexing. 25) One sample at a time: a. Measure the volume of the cell suspension using a pipette. b. Adjust the volume of the cell suspension to 200 µL using base buffer. 26) Acquire the samples on an ID7000 Spectral Cell Analyzer (LE-ID7000C, Sony Biotechnology, USA). .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 68 Supplementary Protocol: Immunohistochemical Staining of Primary Mouse Tumor Sections Day 1 1) Bake the slides with the primary tumor sections at 60°C for at least 1h. 2) Cool the slides to room temperature (RT). 3) Deparaffinize and rehydrate the sections by incubation in xylene and a graded ethanol series: a. Xylene – pass 1 (10 min, RT). b. Xylene – pass 2 (3 min, RT). c. Ethanol (100%) – Pass 1 (3 min, RT). d. Ethanol (100%) – Pass 2 (3 min, RT). e. Ethanol (96%) – Pass 1 (3 min, RT). f. Ethanol (96%) – Pass 2 (3 min, RT). g. Ethanol (80%) (3 min, RT). h. Deionized water (diH2O) (5 min, RT). 4) Move the slides to a container of 1x Dako Target Retrieval Solution, pH 9 (Cat.No. S2367, Agilent, USA). 5) Perform heat-induced epitope retrieval. For example, by a 20-minute incubation in a microwave (Model JT366/WH, Whirlpool, USA) on a no-boil (“6th sense”) setting. 6) Cool the slides in antigen retrieval solution on a benchtop for 10 minutes. 7) Further cool the slides to RT by placing the container of slides in antigen retrieval solution into a sink and pouring room-temperature diH2O into the container. 8) Dry the back of the slides and the area surrounding the tumor section using tissue paper. 9) Outline the tumor sections tightly using a PAP marker (Cat.No. Z627548, Sigma-Aldrich, USA). 10) Place the slides into hydration chambers containing moistened tissue paper. 11) To keep tumor sections hydrated while drying other slides, temporarily add diH2O onto tumor sections on dried slides. 12) Remove excess diH2O from the tumor sections by shaking the slides. 13) Add Dako Real™ Peroxidase Blocking Solution (Cat.No. S2023, Agilent, USA) onto the tumor sections. 14) Incubate for 10 minutes at RT. 15) Wash off the peroxidase blocking solution into a waste container by applying 1x Dako Wash Buffer (Cat.No. S3006, Agilent, USA) using a squeeze bottle. Temporarily leave a small amount of wash buffer on the tumor sections to keep them hydrated while washing other slides. 16) Remove excess wash buffer from the tumor sections by shaking the slides. 17) Add wash buffer onto the tumor sections. 18) Incubate for 5 minutes at RT. 19) Remove the wash buffer from the slides into a waste container. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 69 20) For a second time, add wash buffer onto the tumor sections. 21) Incubate for 5 minutes at RT. 22) Wash off the wash buffer into a waste container by applying diH2O using a squeeze bottle. Sufficient wash buffer has been removed once the surface tension of the liquid on the slide allows the PAP marker outline to become clearly visible. 23) To keep tumor sections hydrated while washing other slides, temporarily add diH2O onto washed tumor sections. 24) Remove excess diH2O from the tumor sections by shaking the slides. 25) Add blocking solution (3% w/V bovine serum albumin (BSA) in phosphate-buffered saline (PBS)) onto the tumor sections. 26) Incubate for 45-60 minutes at room temperature. 27) Wash off the blocking solution into a waste container by applying wash buffer using a squeeze bottle. Temporarily leave a small amount of wash buffer on the tumor sections to keep them hydrated while washing other slides. 28) Remove excess wash buffer from the tumor sections by shaking the slides. 29) Add wash buffer onto the tumor sections. 30) Incubate for 5 minutes at RT. 31) Remove the wash buffer from the tumor sections into a waste container. 32) For a second time, add wash buffer onto the tumor sections. 33) Incubate for 5 minutes at RT. 34) Wash off the wash buffer into a waste container by applying diH2O using a squeeze bottle. Sufficient wash buffer has been removed once the surface tension of the liquid on the slide allows the PAP marker outline to become clearly visible. 35) To keep tumor sections hydrated while washing other slides, temporarily add diH2O onto washed tumor sections. 36) One slide at a time, remove as much excess diH2O as possible, avoiding damaging the tumor sections, then apply the appropriate primary antibody (diluted in 0.5% w/V BSA) to the tumor sections. 37) Incubate tumor sections in primary antibody overnight in a fridge (4°C). .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 70 Day 2 38) Retrieve the primary-antibody-stained tumor sections from the fridge. 39) Wash off the primary antibody solutions by tilting the slide over a waste container and applying wash buffer using a squeeze bottle. 40) Immediately place washed slides into a container of wash buffer. 41) Incubate on an orbital shaker for 5 minutes at RT. 42) Move the slides into a fresh container of wash buffer. 43) Incubate on an orbital shaker for 5 minutes at RT. 44) Wash off the wash buffer into a waste container by applying diH2O using a squeeze bottle. Sufficient wash buffer has been removed once the surface tension of the liquid on the slide allows the PAP marker outline to become clearly visible. 45) To keep tumor sections hydrated while washing other slides, temporarily add diH2O onto washed tumor sections. 46) Remove excess diH2O from the tumor sections by shaking the slides. 47) Apply the appropriate horseradish-peroxidase-conjugated secondary antibody (e.g. Dako EnVision+ System-HRP Labelled Polymer Anti-Mouse (Cat.No. K4001, Agilent, USA) or Dako EnVision+ System- HRP Labelled Polymer Anti-Rabbit (Cat.No. K4003, Agilent, USA)) to the tumor sections. 48) Incubate the tumor sections in secondary antibody for 30 minutes at RT. 49) Wash off the secondary antibody solutions by tilting the slide over a waste container and applying wash buffer using a squeeze bottle. 50) Immediately place washed slides into a container of wash buffer. 51) Incubate on an orbital shaker for 5 minutes at RT. 52) Move the slides into a fresh container of wash buffer. 53) Incubate on an orbital shaker for 5 minutes at RT. 54) Wash off the wash buffer into a waste container by applying diH2O using a squeeze bottle. Sufficient wash buffer has been removed once the surface tension of the liquid on the slide allows the PAP marker outline to become clearly visible. 55) To keep tumor sections hydrated while washing other slides, temporarily add diH2O onto washed tumor sections. 56) Remove excess diH2O from the tumor sections by shaking the slides. 57) Apply a solution of diaminobenzidine (Liquid DAB+, 2-component system (Cat.No. K3468, Agilent, USA)) to the tumor sections. Exercise caution while handling and disposing of diaminobenzidine due to its toxicity. 58) Incubate tumor sections in diaminobenzidine solution in the dark for 8 minutes at RT. 59) Thoroughly wash off the diaminobenzidine solution by tilting the slide over a toxic waste container and applying diH2O using a squeeze bottle. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint 71 60) To keep tumor sections hydrated while washing other slides, temporarily add diH2O onto washed tumor sections. 61) Remove excess diH2O from the tumor sections by shaking the slides. 62) Place the slides into a container of hematoxylin (Cat.No. S3301, Agilent, USA). 63) Incubate the tumor sections in hematoxylin for 10 minutes at RT. 64) Remove the slides from the hematoxylin and shake off the excess. 65) Place the slides into an empty container and wash the remaining hematoxylin off using several portions of warm tap water. 66) Place the slides into a container of diH2O. 67) Dehydrate the tumor sections and prepare them for mounting by dipping them 10 times into each container of a graded ethanol series and xylene: a. Ethanol (80%) b. Ethanol (96%) – Pass 1 c. Ethanol (96%) – Pass 2 d. Ethanol (100%) – Pass 1 e. Ethanol (100%) – Pass 2 f. Xylene – pass 1 g. Xylene – pass 2 68) Mount the slides. .CC-BY 4.0 International licenseavailable under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made The copyright holder for this preprintthis version posted August 11, 2025. ; https://doi.org/10.1101/2025.08.08.669244doi: bioRxiv preprint