Abstract p53 is a transcription factor that plays a central role in guarding the genomic stability of cells through cell-cycle arrest or induction of apoptosis. However, the effects of p53 in antitumor immunity are poorly understood. To investigate the role of p53 in controlling tumor-immune cell cross-talk, we studied murine syngeneic models treated with HDM201, a potent and selective second-generation MDM2 inhibitor. In response to HDM201 treatment, the percentage of dendritic cells increased, including the CD103+ antigen cross-presenting subset. Furthermore, HDM201 increased the percentage of Tbet+Eomes+ CD8+ T cells and the CD8+/Treg ratio within the tumor. These immunophenotypic changes were eliminated with the knockout of p53 in tumor cells. Enhanced expression of CD80 on tumor cells was observed in vitro and in vivo, which coincided with T-cell–mediated tumor cell killing. Combining HDM201 with PD-1 or PD-L1 blockade increased the number of complete tumor regressions. Responding mice developed durable, antigen-specific memory T cells and rejected subsequent tumor implantation. Importantly, antitumor activity of HDM201 in combination with PD-1/PD-L1 blockade was abrogated in p53-mutated and knockout syngeneic tumor models, indicating the effect of HDM201 on the tumor is required for triggering antitumor immunity. Taken together, these results demonstrate that MDM2 inhibition triggers adaptive immunity, which is further enhanced by blockade of PD-1/PD-L1 pathway, thereby providing a rationale for combining MDM2 inhibitors and checkpoint blocking antibodies in patients with wild-type p53 tumors. Significance: This study provides a mechanistic rationale for combining checkpoint blockade immunotherapy with MDM2 inhibitors in patients with wild-type p53 tumors.
Abstract We recently developed ‘Diffuse in vivo Flow Cytometry’ (DiFC), a new pre-clinical research tool for enumerating extremely rare fluorescently-labeled circulating cells directly in vivo . In this paper, we developed a green fluorescent protein (GFP) compatible version of DiFC, and used it to non-invasively monitor the circulating tumor cell (CTC) burden over time in a multiple myeloma disseminated xenograft model. We show that DiFC allowed counting of CTCs at estimated concentrations below 1 cell per mL in peripheral blood with a negligible false alarm rate. DiFC also revealed the presence of CTC clusters in circulation to our knowledge for the first time in this model, and allowed us to calculate their size, kinetics, and frequency of shedding. We anticipate that the unique capabilities of DiFC will have many applications in the study of hematogenous metastasis, and as a powerful complementary methodology to liquid biopsy assays.
<div>Abstract<p>p53 is a transcription factor that plays a central role in guarding the genomic stability of cells through cell-cycle arrest or induction of apoptosis. However, the effects of p53 in antitumor immunity are poorly understood. To investigate the role of p53 in controlling tumor-immune cell cross-talk, we studied murine syngeneic models treated with HDM201, a potent and selective second-generation MDM2 inhibitor. In response to HDM201 treatment, the percentage of dendritic cells increased, including the CD103<sup>+</sup> antigen cross-presenting subset. Furthermore, HDM201 increased the percentage of Tbet<sup>+</sup>Eomes<sup>+</sup> CD8<sup>+</sup> T cells and the CD8<sup>+</sup>/Treg ratio within the tumor. These immunophenotypic changes were eliminated with the knockout of p53 in tumor cells. Enhanced expression of CD80 on tumor cells was observed <i>in vitro</i> and <i>in vivo</i>, which coincided with T-cell–mediated tumor cell killing. Combining HDM201 with PD-1 or PD-L1 blockade increased the number of complete tumor regressions. Responding mice developed durable, antigen-specific memory T cells and rejected subsequent tumor implantation. Importantly, antitumor activity of HDM201 in combination with PD-1/PD-L1 blockade was abrogated in p53-mutated and knockout syngeneic tumor models, indicating the effect of HDM201 on the tumor is required for triggering antitumor immunity. Taken together, these results demonstrate that MDM2 inhibition triggers adaptive immunity, which is further enhanced by blockade of PD-1/PD-L1 pathway, thereby providing a rationale for combining MDM2 inhibitors and checkpoint blocking antibodies in patients with wild-type p53 tumors.</p>Significance:<p>This study provides a mechanistic rationale for combining checkpoint blockade immunotherapy with MDM2 inhibitors in patients with wild-type p53 tumors.</p></div>
We used a folate-receptor targeted fluorescent molecular probe to label circulating tumor cells directly in the bloodstream in mouse metastasis models. We used a new diffuse light-based optical instrument to non-invasively detect and count them.
<p>Supplementary information containing six supplementary figures (Figure S1-S6), one supplementary table (Table S1) and supplementary materials and methods. Supplementary Figure S1. Flow cytometric gating strategies for analysis of intra-tumoral T cells in parental and p53KO Colon26 tumors. Supplementary Figure S2. Flow cytometric gating strategies for analysis of intra-tumoral myeloid cells in parental and p53KO Colon26 tumors. Supplementary Figure S3. Anti-tumor activity of HDM201 in Colon26 syngeneic tumor model requires intact immune system. Supplementary Figure S4. p53 knockout in Colon 26 cells results in loss of response to HDM201 in vitro and in vivo. Supplementary Figure S5. HDM201 in combination with PD-1/PD-L1 blockade enhances anti-tumor responses in p53 wild-type tumors, but not in KO tumors. Supplementary Figure S6. Flow cytometric analysis of CD80, GITR, PD-L1, and CD86 expression after HDM201 treatment in vitro. Supplementary Table S1. Antibodies for flow cytometry analysis.</p>
Abstract Circulating tumor cells (CTCs) are of great interest in cancer research, but methods for their enumeration remain far from optimal. We developed a new small animal research tool called “Diffuse in vivo Flow Cytometry” (DiFC) for detecting extremely rare fluorescently-labeled circulating cells directly in the bloodstream. The technique exploits near-infrared diffuse photons to detect and count cells flowing in large superficial arteries and veins without drawing blood samples. DiFC uses custom-designed, dual fiber optic probes that are placed in contact with the skin surface approximately above a major vascular bundle. In combination with a novel signal processing algorithm, DiFC allows counting of individual cells moving in arterial or venous directions, as well as measurement of their speed and depth. We show that DiFC allows sampling of the entire circulating blood volume of a mouse in under 10 minutes, while maintaining a false alarm rate of 0.014 per minute. In practice, this means that DiFC allows reliable detection of circulating cells below 1 cell per mL. Hence, the unique capabilities of DiFC are highly suited to biological applications involving very rare cell types such as the study of hematogenous cancer metastasis.
An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures.
Abstract Purpose We recently developed a new instrument called ‘diffuse in vivo flow cytometry’ (DiFC) for enumeration of rare fluorescently-labeled circulating tumor cells (CTCs) in small animals without drawing blood samples. Until now, we have used cell lines that express fluorescent proteins, or were pre-labeled with a fluorescent dye ex-vivo . In this work, we investigated the use of two folate receptor (FR)-targeted fluorescence molecular probes for in vivo labeling of FR+ CTCs for DiFC. Methods We used EC-17 and Cy5-PEG-FR fluorescent probes. We studied the affinity of these probes for L1210A and KB cancer cells, both of which over-express FR. We tested the labeling specificity in cells in culture in vitro , in whole blood, and in mice in vivo . We also studied detectability of labeled cells with DiFC. Results Both EC-17 and Cy5-PEG-FR probes had high affinity for FR+ CTCs in cell culture in vitro . However, only EC-17 had sufficient specificity for CTCs in whole blood. EC-17 labeled CTCs were also readily detectable in circulation in mice with DiFC. Conclusions This work demonstrates the feasibility of labeling CTCs for DiFC with a cell surface receptor targeted probe, greatly expanding the utility of the method for pre-clinical animal models. Because DiFC uses diffuse light, this method could be also used to enumerate CTCs in larger animal models and potentially even in humans.
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