Abstract 4704: Development of a novel zebrafish patient-derived xenograft (zPDX) platform for functional precision cancer medicine for immune checkpoint (ICIs) and tyrosine kinase inhibitors (TKIs) of renal cell carcinoma (RCC)

Abstract

Abstract Predictions of cancer treatment outcomes are critical to personalized therapy. Despite extensive studies of genome-based precision medicine, we still lack reliable markers to predict patient-specific response to ICIs and TKIs for RCC. zPDX has been used to develop in vivo preclinical models using mouse PDX, primary cultured cells or dissociated tissues (without T cell analysis). These samples do not reflect enough of the intratumor heterogeneity. To create assays better representative of their natural state, we used freshly isolated tumor cells for zPDX and tested their ability to display patient-specific response to ICIs and TKIs. Within 24h after nephrectomy, the RCC mass (0.8-1.9 g) was extracted and purified for whole tumor cells (WTCs) via Ficoll gradient centrifugation and dead cell removal (viability >85%; yield 0.3-33 x 106 cells) and analyzed by FACS for % CD45+ or CD8+ cells (or PD1+CD8+). RCC-WTC was rich in CD45+ immune cells (65% ± 10, n=8) and CD8+ T cells (38% ± 10, n=5), among which PD1 expression was low (4.7% ± 2.2, n=5). WTCs were labeled with Red Cell-Tracker, mixed with Nivolumab or control IgG (0.5 ng/fish), injected into the perivitelline space (PVS) of 2dpf zebrafish embryos (∼200 cells/fish, n=10/group), and zPDX cultured individually in a microwell for 2-5d at 34°C. For TKIs, WTC-injected zebrafish were cultured in media with Cabozantinib (0.1 μM) or Sunitinib (0.3 μM) (n=8/group). 2-4d post-injection, fluorescence images were taken and analyzed for tumor area (TA; procedural defined units) around PVS. % reduction in TA was recorded as response and RECIST-categorized into PR (>30% reduction), PD (>30% increase) and SD (<30% reduction). To date, we have performed zPDX assays for 8 cases (none with pre-determined treatment plans). For ICIs, 6 cases displayed PR (53% ± 1.6, p<0.003) and the rest (2 cases) did SD (11% ± 10) with weak association of ICI-effects with PD1 expression, but PR responders (5 out of 6) showed significant upregulation of hIL-2 mRNA (median 6.2 x10-5 to hGAPDH) by nested qRT-PCR of total RNA from Nivolumab-treated zPDX. This tumor inhibition was T cell-mediated, shown by co-applied anti-CD3 Ab that reversed tumor regression by ICI (n=3, 82-100% reversal) and the presence of hT-cells in zPDX treated with Nivolumab (but not with control IgG) shown by IHC staining of whole body FFPE with anti-CD3. TKIs inhibited TA, with 50% PR response rate for Sunitinib and Cabozantinib (38% ± 5.5, p<0.05 & 40% ± 7.7, p<0.02, respectively) with significant individual differences in % inhibition. We showed our zPDX model with freshly isolated WTC can quantify patient-specific responses to ICIs and TKIs before onset of actual treatments. Thus, this zPDX platform will establish a fast, accurate, and clinically relevant system for functional precision medicine for RCC immunotherapy. Citation Format: Nilambari Pawar, Jin-Sung Chung, Victoria Cantrell, Irene Dougherty, Nicholas Bingham, Vitaly Margulis, Ponciano D. Cruz, Hans Hammers, Kiyoshi Ariizumi. Development of a novel zebrafish patient-derived xenograft (zPDX) platform for functional precision cancer medicine for immune checkpoint (ICIs) and tyrosine kinase inhibitors (TKIs) of renal cell carcinoma (RCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4704.

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