Kathryn J. Brayer

Survival time is reduced in huNBSGW PDX models. A, huNBSGW PDX3, 9, and 18 were engrafted with the same pooled donor CD34+ cells. Survival time was PDX dependent with PDX3 and 9 having a shorter survival time compared with PDX18. Survival curves are significantly different between huPDX and non-huPDX for all three PDX models (P < 0.025, log-rank test). For all PDX models, n = 3 mice/group for huPDX and non-huPDX. B, Solid tumor mass (in grams) of huPDX and non-huPDX at necropsy. Bars are the average tumor burden, error bars are SEM. Measures for individual mice are shown as black dots (n = 2–3 per group). C, Ascites fluid volume at necropsy. The ascites fluid volume trended higher in non-huPDX for PDX3 and PDX9, but was only significantly different for PDX9. Statistical analysis for B and C used unpaired t tests to compare huPDX versus non-huPDX values for each PDX line.

Cytokines elevated in the ascites fluid of patients with ovarian cancer are also present in the huPDX ascites fluid

HALO analysis of tumor vascularization. A, The levels of VEGF-A in PDX ascites fluid was measured by ELISA. B, To quantify tumor vascularization in huPDX versus non-huPDX tumors, vessels were labeled with anti-CD31 antibody and vessel area was determined by analyzing scanned slides. The tumor tissue was masked on the basis of H&E staining (green) and CD31 labeled vessels were detected (red). Vessel density was calculated using the tissue classifier analysis software on the HALO platform. C, The quantification of vascular density (% vascular area) across whole sections of huPDX and non-huPDX samples. Values are the average vascular densities among samples (n = 2–3). huPDX18 sections showed increased vascular density compared to non-huPDX18 (P < 0.015, unpaired t test). D and E, Comparison of vascularity in representative images from huPDX18 (D) and non-huPDX18 (E) labeled with anti-CD31 antibody. Scale bar, 200 μm.

HALO analysis of tumor vascularization. A, The levels of VEGF-A in PDX ascites fluid was measured by ELISA. B, To quantify tumor vascularization in huPDX versus non-huPDX tumors, vessels were labeled with anti-CD31 antibody and vessel area was determined by analyzing scanned slides. The tumor tissue was masked on the basis of H&E staining (green) and CD31 labeled vessels were detected (red). Vessel density was calculated using the tissue classifier analysis software on the HALO platform. C, The quantification of vascular density (% vascular area) across whole sections of huPDX and non-huPDX samples. Values are the average vascular densities among samples (n = 2–3). huPDX18 sections showed increased vascular density compared to non-huPDX18 (P < 0.015, unpaired t test). D and E, Comparison of vascularity in representative images from huPDX18 (D) and non-huPDX18 (E) labeled with anti-CD31 antibody. Scale bar, 200 μm.

High M-CSF production influences the development of myeloid cells in huNBSGW PDX. A, Diagram of the huPDX study. B, Change in human CD45+ cell populations in peripheral blood before tumor engraftment versus end stage for each mouse (n = 3 mice/group). CD19+ B cells, CD3+ T cells, and CD11b+ myeloid cells make up the majority of human immune cells in the blood. Note that terminal samples from huPDX9 ms3 were not collected as the mouse died unexpectedly overnight. Mouse numbers are listed as ms1–3. C, Average percentage of human myeloid cells in the peripheral blood of huPDX before and after tumor challenge (values are the average ± SD). P values are based on an unpaired two-tailed Student t test. ***, P < 0.0005; **, P < 0.005; *, P < 0.05; n.s., not significant). D, Percentage of CD11b+ human myeloid cells in huPDX ascites fluid at end stage. E, M-CSF concentration in patient ascites fluid measured by ELISA. Samples were assayed in duplicate. Error bars are SD. F, M-CSF concentrations in PDX ascites fluid measured by cytokine array. G, M-CSF gene expression levels in patient samples as determined by RNA-seq analysis of primary patient samples and non-huPDX samples. H, GMCSF concentrations in PDX ascites fluid measured by cytokine array. Black dots represent the value for each sample. Bars are the average level and error bars are SEM. H = humanized NH = non-humanized.

Detection of TAMs in huNBSGW PDX tumor sections. A, Serial sections of huNBSGW PDX tumor tissue were labeled with human-specific antibodies against a mitochondrial protein to label all human cells and the TAM marker CD68. Scale bar, 100 μm. B, Quantification of the number of TAMs using HALO software to identify CD68+ cells. Values are no. of positive cells/100,000 cells. The box plots indicate the mean, minimum, and maximum. Black dots indicate values for each tumor analyzed (n = 2–3).

Analysis of human cytokine levels in huPDX and non-huPDX models indicates their origin. A–C, Examples of the subtypes of cytokines in huPDX. A, PDX-intrinsic cytokines. B, Immune cell–dependent cytokines. C, Immune cell–influenced cytokines. D–F, Tables of cytokine levels in ascites fluid. Levels are presented as the natural log of the least squares means (non-huPDX n = 7, huPDX n = 7) D. PDX-intrinsic cytokines (levels >1 in non-huPDX). Factors with detectable levels in only one or two PDX models are listed separately. E, Immune cell–dependent cytokines with non-huPDX levels <1. F, Factors with detectable levels in the non-huPDX, but significantly increased levels in the huPDX.

Detection of Human TILs in huNBSGW PDX tumor sections. A, Tumor sections from huPDX were labeled for human T-cell markers CD3, CD4, and CD8. Shown are scanned images of labeled sections at 20× magnification. Representative images are shown for huPDX3 and huPDX18. For huPDX9, the image shown is a rare group of TILs in a mostly negative section to show that the rare T cells were being labeled. Scale bar, 100 μm. B, Quantification of the number of TILs using HALO software. Values are reported as no. of positive cells/100,000 cells. The box plots indicate the mean, minimum, and maximum. Black dots indicate values for each tumor analyzed (n = 2–3).

High M-CSF production influences the development of myeloid cells in huNBSGW PDX. A, Diagram of the huPDX study. B, Change in human CD45+ cell populations in peripheral blood before tumor engraftment versus end stage for each mouse (n = 3 mice/group). CD19+ B cells, CD3+ T cells, and CD11b+ myeloid cells make up the majority of human immune cells in the blood. Note that terminal samples from huPDX9 ms3 were not collected as the mouse died unexpectedly overnight. Mouse numbers are listed as ms1–3. C, Average percentage of human myeloid cells in the peripheral blood of huPDX before and after tumor challenge (values are the average ± SD). P values are based on an unpaired two-tailed Student t test. ***, P < 0.0005; **, P < 0.005; *, P < 0.05; n.s., not significant). D, Percentage of CD11b+ human myeloid cells in huPDX ascites fluid at end stage. E, M-CSF concentration in patient ascites fluid measured by ELISA. Samples were assayed in duplicate. Error bars are SD. F, M-CSF concentrations in PDX ascites fluid measured by cytokine array. G, M-CSF gene expression levels in patient samples as determined by RNA-seq analysis of primary patient samples and non-huPDX samples. H, GMCSF concentrations in PDX ascites fluid measured by cytokine array. Black dots represent the value for each sample. Bars are the average level and error bars are SEM. H = humanized NH = non-humanized.