Single-agent checkpoint blockade inhibitors have shown great promise in recent years but has unfortunately been limited to a subset of tumor types. The tumor mutation burden as well as the tumor microenvironment are two key important features that distinguish immunogenic, from non-immunogenic tumors. There is currently a massive effort, including a wide range of strategies, turning tumors from cold to hot including combining with small molecule inhibitors. For colorectal (CRC) cancers, anti-PD1 therapies have proven to be successful in the clinic for microsatellite instable (MSI-high) tumors but have no efficacy in the majority of patients with CRC that have microsatellite stable (MSS) cancers. Our primary goal is to determine if the addition of binimetinib (small molecule MEK inhibitor) and bevacizumab (anti-VEGF therapeutic antibody) increases the response rate of metastatic microsatellite stable colorectal cancer to pembrolizumab in humanized patient-derived xenografts (hPDXs) of CRC. We will also identify immune and pathway modulation in MSS CRC hPDXs treated with the combination of MEK, VEGF, and immune checkpoint inhibitors.
Methods
In six independent experiments, we implanted distinct MSS CRC PDXs, that were recently isolated from patients on a matching clinical trial, into the flanks of humanized BRGS (BALB/c, Rag2-/-, IL2RgC-/-, NODSIRPa) mice that had been engrafted with human hematopoietic stem cells at birth. For each PDX we generated humanized mice cohorts treated with vehicle, binimetinib, binimetinib/pembrolizumab combination, or binimetinib/pembroliumab/bevacizumab/DC101. The human immune system in the immune organs and tumors were interrogated by flow cytometry to assess changes in the cellular composition and the activation state of the immune system as a result of treatments, and the expression of immune-related molecules were assessed on the tumor cells.
Results
There were no significant differences in primary tumor growth in all treated models. However, immune modulation was observed in TILs in which we measured increased activated T cells (DR+, effector memory, TIM-3+), GrB+CD8+ and IFNg+CD8+ T cells. We also observed increased TNFa and IFNg and decreased T regulatory (FoxP3+CD25+) CD4+ T cells in the pembrolizumab/binimetinib and triple combination groups. Immune infiltrates were unique for the various PDXs.
Conclusions
In this preclinical examination of combination MEK, VEGF, and PD-1 inhibition in CRC hPDXs no significant differences in tumor growth were noted, but immune modulation in TILs and tumor were observed suggesting potential immune modulation of the tumor microenvironment that may lead to greater susceptibility to immune checkpoint inhibition in patients with MSS mCRC.
Ethics Approval
The human cord blood samples were generously provided as de-identified donors from Clinimmune Cord Blood Bank (Aurora, CO). All procedures and mouse husbandry were performed in accordance with IACUC protocols approved by the University of Colorado Denver Institutional Animal Care and Use Committee in the Office of Laboratory of Animal Resources (OLAR), a facility approved by the American Association for Laboratory Animal Care.
Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, "humanized") mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2-/- IL2RγC-/- NODSIRPα) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; "hot" tumors exhibit large immune infiltration while "cold" tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.
