Behavior of CAR-T cells in OvCAR3/FB gels

Vascularized microfluidic chip to investigate CAR-T cell migration and cytotoxicity.

Impaired death receptor signaling in malignant cells caused resistance to CAR-T cell cytotoxicity.

Elevated extracellular matrix (ECM) associates with chemo-resistance and poor prognosis in cancer. Modifying the ECM could enhance response to chemotherapy. We measured chemotherapy-induced changes in the tumor microenvironment (TME) of two high-grade serous ovarian cancer (HGSOC) mouse models with different chemo-sensitivity. Carboplatin/paclitaxel treatment triggered dynamic transcriptional changes in immune and ECM-related pathways, and analyses of the ECM structure revealed modifications in the chemo-sensitive tumors. These changes, observed over twenty days post-chemotherapy, had relevance to HGSOC patient responses. Integrating transcriptomics with ECM structure metrics, we identified ECM targets, including lysyl oxidase (LOX), that might enhance effects of chemotherapy. Given alone or with chemotherapy, a pan-LOX inhibitor, PXS-5505, modulated fibroblast and immune cell distribution and decreased tumor stiffness in the less-responsive mouse model. Moreover, treatment with PXS-5505 before chemotherapy enhanced the therapeutic response. We conclude that pretreatment with ECM targeting agents may improve chemotherapy efficacy by altering ECM structure and immune responses.

Fluorophore-conjugated antibodies used for flow cytometry.

OvCAR3 cells were sensitive but G164 cells were resistant to CAR-T cell cytotoxicity in monolayer cultures.

Luminal flow of CAR-T cells through vessels formed in the microfluidic device

Malignant cells and fibroblasts co-culture collagen gels treated with CAR-T cells.

CCL2 produced by fibroblasts activated CCR2/4+ CAR-T cells to induce antigen-dependent cytotoxicity.

Primary antibodies used for immunohistochemistry.

Behavior of CAR-T cells in G164/FB gels without TGFβ-R inhi

Microvasculature formed within fibrin gel penetrated the OvCAR3 collagen gel

CCL2 produced by fibroblasts activated CCR2/4+ CAR-T cells to induce antigen-dependent cytotoxicity.

High-grade serous ovarian cancer is generally treated with upfront chemotherapy, including carboplatin. The persistence of platinum-resistant cells drives recurrent disease. A high-throughput screen using a 3D organotypic culture assembled with extracellular matrix, primary human fibroblasts, and mesothelial cells was established and validated. Using a library of FDA-approved drugs, the 3D high-throughput screen was performed with the goal of identifying a combination of drugs that synergistically target two populations of ovarian cancer: aldehyde dehydrogenase (ALDH) high (ALDHhi) and ALDH low (ALDHlo) enzyme activity cells, which are less sensitive to carboplatin treatment than the bulk ovarian cancer cells. Initial results showed that omipalisib, verteporfin, CA3, mitoxantrone, navitoclax, venetoclax, and YM155 had significant single-drug activity in either the ALDHlo or both the ALDHlo/ALDHhi cell populations. Synergistic drug activity was identified with three drug combinations: navitoclax/omipalisib, navitoclax/YM155, and YM155/omipalisib. In vitro, the combination of navitoclax/YM155 was most efficient at blocking primary human ovarian cancer sphere formation and the proliferation of four different ovarian cancer cell lines in the 3D organotypic culture. In vivo, the combination of navitoclax/YM155/carboplatin decreased ovarian cancer metastasis, decreased the percentage of ALDHhi ovarian cancer cells in tumors, and increased survival when compared with carboplatin treatment alone in xenograft models. Our results suggest that the combination of navitoclax/YM155/carboplatin has promise as a therapy for treating ovarian cancer.

CCL2 produced by fibroblasts activated CCR2/4+ CAR T cells to induce antigen-dependent cytotoxicity. A, Cytokine profile of FB-, G164-, and OvCAR3-conditioned medium using proteome profiler array. FBs from two donors were used for this experiment. Each cytokine is in duplicate. Dots that are not labeled are reference points. B, ELISA showing CCL2 concentrations in monolayers and spheroids of malignant cells with and without FB. Two FB donors were used for this experiment. Data plotted as mean ± SD of two replicates. C and D, IFNγ concentration measured by ELISA (C) and Incucyte CAR T-cell killing assay of G164 spheroids with no treatment (NT) or with recombinant CCL2 (D). E and F, ELISA data showing IFNγ concentration (E) and Incucyte killing assay of G164 spheroids cultured in FB-M with anti-CCL2 (aCCL2) antibody (F). G, Fold change analysis of the expression of T-cell activation markers HLA-DR and CD69 on CCR2+ (top) and CCR4+ (bottom) CAR T cells after CCL2, FB-M, or FB-M with aCCL2 treatments. C, E, and G, Data plotted as mean ± SD for three CAR T-cell donors. Statistics performed using two-way (C and E) and one-way ANOVA (G). D and F, Data shown for one CAR T-cell donor. Representative images at day 3 (left) and quantification (right). Red, dead cells. Scale bar, 400 μm.

Studying cell behavior in live human tumors is crucial to understand and improve response to immunotherapies. Here, we present a protocol to slice human ovarian tumors ex vivo , maintain their viability for 24 h, and monitor the behavior of CD8 + T and myeloid cells in real time. Furthermore, we detail procedures to semi -automatically analyze cell movements and aggregate and process behavior data. This protocol can potentially be applied for multiple tumor types and mouse cancer models. For complete details on the use and execution of this protocol, please refer to Laforets et al. 1

Abstract In vitro preclinical testing of chimeric antigen receptor (CAR) T cells is mostly carried out in monolayer cell cultures. However, alternative strategies are needed to take into account the complexity and the effects of the tumor microenvironment. Here, we describe the modulation of CAR T-cell activity by malignant cells and fibroblasts in human three-dimensional (3D) in vitro cell models of increasing complexity. In models combining mucin-1 (MUC1) and TnMUC1 CAR T cells with human high-grade serous ovarian cancer cell spheroids, malignant cell–intrinsic resistance to CAR T-cell killing was due to defective death receptor signaling involving TNFα. Adding primary human fibroblasts to spheroids unexpectedly increased the ability of CAR T cells to kill resistant malignant cells as CCL2 produced by fibroblasts activated CCR2/4+ CAR T cells. However, culturing malignant cells and fibroblasts in collagen gels engendered production of a dense extracellular matrix that impeded CAR T-cell activity in a TGFβ-dependent manner. A vascularized microfluidic device was developed that allowed CAR T cells to flow through the vessels and penetrate the gels in a more physiological way, killing malignant cells in a TNFα-dependent manner. Complex 3D human cell models may provide an efficient way of screening multiple cytotoxic human immune cell constructs while also enabling evaluation of mechanisms of resistance involving cell–cell and cell–matrix interactions, thus accelerating preclinical research on cytotoxic immune cell therapies in solid tumors. Significance: Three-dimensional in vitro models of increasing complexity uncover mechanisms of resistance to CAR T cells in solid tumors, which could help accelerate development of improved CAR T-cell constructs.

Studying cell behavior in live human tumors is crucial to understand and improve response to immunotherapies. Here, we present a protocol to slice human ovarian tumors ex vivo, maintain their viability for 24 h, and monitor the behavior of CD8+ T and myeloid cells in real time. Furthermore, we detail procedures to semi-automatically analyze cell movements and aggregate and process behavior data. This protocol can potentially be applied for multiple tumor types and mouse cancer models.For complete details on the use and execution of this protocol, please refer to Laforets et al.1