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Abstract
Background Rhesus macaques share extensive similarities with humans including immune cell populations, regulatory mechanisms, and targets for cancer immunotherapy, but also naturally develop cancers at similar life stages. Treating rhesus macaques with spontaneous tumors bears enormous potential as a model for human cancer immunotherapy.
Methods For this study (figure 1), we recruited 2 female rhesus macaques with naturally-occurring colorectal cancer (CRC): 23.6y (CRC_1) and 20.3y (CRC_2) old. We collected a pre-treatment tumor biopsy in CRC_1 prior to treatment with 8 Gy intensity-modulated radiotherapy (IMRT) to the proximal portion of the tumor and 2 mg/kg i.v. pembrolizumab. The second candidate (CRC_2) was not tumor biopsy-eligible and treated with pembrolizumab alone. Sampling consisted of blood and peripheral lymph nodes (LNs) pre- and on-treatment and complete tissue collection at the 3w endpoint. Follow-up analysis consisted of flow cytometry (PBMCs), IHC, and transcriptomics.
Results Morphologically, both tumors presented with focal thickening of the proximal colonic wall, and were mismatch repair deficiency. Flow cytometry of PBMCs revealed a strong response of CD4 and CD8 in aPD1+IMRT-treated CRC_1, acutely driven by TEMRA (CD45RA+CD62L-) at 3d and replaced by the increasing Tem population (CD45RA- CD62L-) at the later time points. This was accompanied by an increase of OX40+ (2w, strongest in CD4) and PD1+ T cells (3w, strongest in CD8). aPD1-only CRC_2 presented with a much milder PBMC response, only limited expansion of Tem, a reduced OX40+ upregulation, and no elevation in PD1+ towards the 3w endpoint.
IHC revealed a mild treatment response composed of a reduction of CD4/FoxP3, and increase of high endothelial venules (MECA-79) was observed in peripheral LNs of CRC_1. Additionally, we observed an initial reduction of CD3, CD8, and CD20 staining in peripheral LNs of CRC_2 at 1w, followed by a mild expansion at the later time point. Following therapy, extensive numbers of TLS were observed adjacent to both CRCs, consisting primarily of CD20+ B cells with germinal centers (Ki67+ and PD1+ cells), and T cell zones (CD3+/CD4+/CD4+FoxP3+/CD8+). Both tumors were depleted of CD3+ T cells, particularly in more poorly differentiated regions. Notably, IBA1+ macrophages extensively infiltrated both tumors and were enriched at the tumor margins and the TLS periphery.
Conclusions In rhesus CRCs, we observed similar immune-depleted phenotypes and response to aPD1±IMRT as described in humans, enabling an unprecedented opportunity to dissect the impact of the tumor microenvironment on the acute and long-term response and resistance.
Ethics Approval All procedures were approved by the WFSM Institutional Animal Care and Use Committee, in compliance with the U.S. Animal Welfare Act, The Guide for the Care and Use of Laboratory Animals, the Office of Laboratory Animal Welfare, and Public Health Service Policy. WFSM is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, International (AAALACi).
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