The lack of suitable models currently hampers our understanding of how the tumor microenvironment responds to immunotherapy treatment. Here, we present a pro-tocol for ex vivo culture of patient... Show moreThe lack of suitable models currently hampers our understanding of how the tumor microenvironment responds to immunotherapy treatment. Here, we present a pro-tocol for ex vivo culture of patient-derived tumor fragments (PDTFs). We describe the steps for tumor collection, generation and cryopreservation of PDTFs, and their subsequent thawing. We detail culture of PDTFs and their preparation for analysis. This protocol preserves the tumor microenvironment's composition, architecture, and cellular interactions, which can be perturbed by ex vivo treatment. For complete details on the use and execution of this protocol, please refer to Voabil et al. (2021).1 Show less
Neoadjuvant immunotherapy with anti-cytotoxic T lymphocyte-associated protein 4 (CTLA4) + anti-programmed cell death protein 1 (PD1) monoclonal antibodies has demonstrated remarkable pathological... Show moreNeoadjuvant immunotherapy with anti-cytotoxic T lymphocyte-associated protein 4 (CTLA4) + anti-programmed cell death protein 1 (PD1) monoclonal antibodies has demonstrated remarkable pathological responses and relapse-free survival in -80% of patients with clinically detectable stage III melanoma. However, about 20% of the treated patients do not respond. In pretreatment biopsies of patients with melanoma, we found that resistance to neoadjuvant CTLA4 + PD1 blockade was associated with a low CD4/interleukin-2 (IL-2) gene signature. Ex vivo, addition of IL-2 to CTLA4 + PD1 blockade induced T cell activation and deep immunological responses in anti-CTLA4 + anti-PD1-resistant human tumor specimens. In the 4T1.2 breast cancer mouse model of neoadjuvant immunotherapy, triple combination of anti-CTLA4 + anti-PD1 + IL-2 cured almost twice as many mice as compared with dual checkpoint inhibitor therapy. This improved efficacy was due to the expansion of tumor-specific CD8(+) T cells and improved proinflammatory cytokine polyfunctionality of both CD4(+) and CD8(+) T effector cells and regulatory T cells. Depletion studies suggested that CD4(+) T cells were critical for priming of CD8(+) T cell immunity against 4T1.2 and helped in the expansion of tumor-specific CD8(+) T cells early after neoadjuvant triple immunotherapy. Our results suggest that the addition of IL-2 can overcome resistance to neoadjuvant anti-CTLA4 +anti-PD1, providing the rationale for testing this combination as a neoadjuvant therapy in patients with early-stage cancer. Show less
Voabil, P.; Bruijn, M. de; Roelofsen, L.M.; Hendriks, S.H.; Brokamp, S.; Braber, M. van den; ... ; Thommen, D.S. 2021
An ex vivo platform of patient-derived tumor fragments enables the assessment of intratumoral immune reactivation after PD-1 blockade that is predictive of clinical outcomes in patients with cancer... Show moreAn ex vivo platform of patient-derived tumor fragments enables the assessment of intratumoral immune reactivation after PD-1 blockade that is predictive of clinical outcomes in patients with cancer.Inhibitors of the PD-1-PD-L1 axis have been approved as therapy for many human cancers. In spite of the evidence for their widespread clinical activity, little is known about the immunological alterations that occur in human cancer tissue after PD-1 blockade. We developed and employed a patient-derived tumor fragment platform to dissect the early immunological response of human tumor tissue to ex vivo PD-1 blockade. We observed that the capacity of immune cells to be reactivated ex vivo was predictive of clinical response, and perturbation analyses identified tumor-resident T cells as a key component of this immunological response. In addition, through combined analysis of baseline properties and immune response capacity, we identified a new subgroup of infiltrated tumors that lacks the capacity to respond to PD-1 blockade. Finally, the baseline presence of tertiary lymphoid structures and their components correlated with the capacity of cancers to undergo intratumoral immune cell reactivation. Show less
