PMC9659479_01
Patient with primary RCC. (A) CT scan of abdomen with sagittal view showed a 13 cm right renal solid, enhancing mass with invasion into the liver and left hepatic lobe metastatic lesions. The blue arrows indicate tumor involvement. (B,C) Histological evaluation of resected primary specimen (B: x10; C: x40) showed clear cell component. (D) Generation of RCC PDX mice. The resected nephrectomy specimen was minced and injected subcutaneously into 8-10 weeks old NSG mice. Following injection, the site of injection was monitored until a tumor was observed (approximately 8 weeks). Tumors were then measured 2-3 times per week until tumors reached ~1,000 mm3. Tumors were then harvested and serially re-implanted until a stable RCC PDX was established. (E) CT scan of chest with coronal view before nivolumab treatment. (F) CT scan of chest with coronal view after 60 cycles of nivolumab treatment. RCC, renal cell carcinoma; PDX, patient-derived xenograft; ARGOS, Argos Therapeutics Inc; ADAPT, Phase 3 trial of autologous dendritic cell immunotherapy plus standard treatment of advanced renal cell carcinoma (ClinicalTrials.gov identifier: NCT01582672); CNS, central nervous system; CT, computed tomography; NSG, non-obese diabetic scid gamma null.
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A 68-year-old Caucasian man initially presented in January 2014 to the Duke Cancer Center with gross hematuria and was found to have a 13 cm right renal solid, enhancing mass with metastasis to the liver (Figure 1A). Pathology from this surgery was diagnostic of clear cell renal cell carcinoma (RCC), grade 3 with renal vein invasion (Figure 1B,1C). Tumors were then harvested and serially re-implanted until a stable RCC PDX was established (Figure 1D). Prior to initiating nivolumab, he developed extensive metastatic disease including heavy burden in lung pleura, parenchyma, mediastinal, and hilar nodes in addition to malignant pleural effusions requiring pleural drain placement (Figure 1E). He received 60 cycles total of nivolumab and achieved a complete clinical and radiographic remission (Figure 1F).
Engraftment of human HSPC derived hematopoietic cells in RCC PDX mice. (A) Bone marrow aspirate was performed on the patient and ~30 mL bone marrow aspirates were obtained. Enriched human HSPCs were obtained by incubating bone marrow aspirates with the RosetteSepTM pre-enrichment cocktail containing antibodies recognizing T cell, B cell, myeloid cell and platelet. Purified CD34+ HSPCs were then selected using EasySepTM positive selection cocktail from the enriched HSPCs with an antibody recognizing CD34. Enriched human HSPCs (1x105 cells/mouse, n=5) or CD34+ HSPCs (0.1x105 cells/mouse, n=15) were injected intravenously into 8-10 weeks old NSG mice that were radiated with 150 rad using cesium irradiator within 2 hours after the irradiation. Human hematopoietic cell engraftment was measured at indicated time-points by flow cytometry quantification of human CD45+ cells in the peripheral blood. (B) At the end of PDX experiment, i.e., 13 weeks after HSPC injection, the mice were sacrificed and the human CD45+ cells in the bone marrow were measured by flow cytometry. HSPC, hematopoietic stem/progenitor cell; RCC, renal cell carcinoma; PDX, patient-derived xenograft.
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At 11 weeks post-transplant, human CD45+ cells reconstituted 2.3%+-0.179% (mean +- SD) of total peripheral blood cells in NSG mice receiving enriched human HSPCs, and 0.69%+-0.058% of total peripheral blood cells in NSG mice transplanted with human CD34+ HSPCs (Figure 2A,2B).
Autologous humanized RCC PDX mice responded to nivolumab treatment while non-humanized RCC PDX mice showed no response to nivolumab. (A) Autologous humanized RCC PDX mice. The matched patient PDX was injected subcutaneously into the humanized mice derived from patient's own enriched HSPCs (blue line) or purified human CD34+ HSPCs (red line) at 11 weeks post-transplant. Following injection, the site of injection was monitored until a tumor was observed (approximately 3 weeks). Tumors were then measured 2-3 times per week using a vernier caliper until tumors reached 250 mm3. The mice were then injected with nivolumab (200 microg IP weekly) or control (saline IP weekly). Tumor volumes were measured and compared [*, P<0.05 (Mann-Whitney)]. (B) Non-humanized RCC PDX mice. Non-humanized NSG mice were implanted similarly with patient RCC PDXs. When tumors reached 250 mm3, the mice were injected with nivolumab (200 microg IP weekly) or control (saline IP weekly. Tumor volumes were measured and compared. HSPC, hematopoietic stem/progenitor cell; RCC, renal cell carcinoma; PDX, patient-derived xenograft; IP, intraperitoneal.
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Figure 3 shows that the humanized RCC PDX responded to nivolumab therapy whereas the non-humanized RCC PDX did not respond suggesting that our humanized immune model can potentially be used to study immuno-oncology.